CN112646791A - Mutant and construction method and application thereof - Google Patents

Abstract

The invention provides a mutant, the amino acid sequence of which is as follows (a) or (b): (a) at least one amino acid of the amino acid sequence of the natural L-lysine oxidase is substituted, deleted or added, and the sequence has more than 90% homology with the amino acid sequence; or (b) at least one amino acid of the amino acid sequence of a natural L-lysine oxidase is substituted, deleted or added, and has the same function as the sequence. Compared with the wild L-lysine oxidase, the mutant of the L-lysine oxidase provided by the invention has better heat stability. The L-lysine oxidase mutant obtained by the construction method provided by the invention has better thermal stability, still shows excellent catalytic activity when L-lysine is oxidized at higher temperature, and has higher application potential in the fields of L-lysine biosensor detection, biochemical engineering and the like.

Description

Mutant and construction method and application thereof

Technical Field

The invention relates to the technical field of biology, and particularly relates to a mutant and a construction method and application thereof.

Background

L-lysine, also known as the first limiting amino acid, not only regulates the metabolic balance in the body, but also has important effects on improving the absorption of cereal proteins in the body, improving the dietary nutrition of human beings and promoting the growth and development. Therefore, the L-lysine content is an important index for detection. The traditional physical and chemical detection method has long detection time, high cost and complicated operation process, and the biological catalysis is an efficient and simple method. L-amino acid oxidase is an important oxidoreductase participating in the oxidative metabolism of amino acids in organisms, is mostly flavoprotein, and can catalyze L-amino acid oxidative deamination by using oxygen molecules as electron receptors to generate corresponding keto acid, ammonia (NH3) and hydrogen peroxide (H2O 2). Most of the L-amino acid oxidases found at present have a broad substrate spectrum, and are often interfered by other existing amino acids when catalyzing and oxidizing a certain amino acid. Some L-amino acid oxidases are capable of specifically recognizing a specific amino acid without being interfered by other kinds of amino acids. The L-lysine oxidase can perform specific catalytic reaction with L-lysine, is an important catalyst for life activities, has mild catalytic reaction conditions, single product, low energy consumption and easy separation of the product, and is widely applied to the fields of food, chemical industry, environmental protection, energy and medicine. However, the stability and catalytic activity of the natural L-lysine oxidase are greatly reduced under severe conditions of high temperature, extreme pH value, organic solvent, unnatural substrate, product inhibition and the like, and the application requirement in industrial production is difficult to meet.

The protein engineering is based on the relationship between the structural rule and the biological function of protein molecules, and carries out gene modification or gene synthesis by means of chemistry, physics and molecular biology to modify the existing protein or manufacture a new protein to meet the requirements of human on production and life. Rational design is the most common method in protein engineering, and utilizes computer-aided molecular model combined with site-directed mutagenesis to realize protein function optimization, such as improvement of catalytic activity, thermal stability, acid and alkali resistance, etc. To effectively optimize the thermal stability of proteins, Markus Wys et al proposed the Consenssus Concept in 2001. Different from the conventional rational protein design method based on the precise structure-function relationship of protein, the Consensus Concept is based on the amino acid sequence information of homologous protein, and the information capable of improving the thermal stability of enzyme is analyzed from the evolutionary point of view. The invention takes the Consensus theory as a guiding idea, integrates and analyzes the L-amino acid oxidase family sequence, and combines the assistance of bioinformatics to obtain the novel L-lysine oxidase mutant with high stability. Related studies are not reported at present.

Disclosure of Invention

Therefore, the present invention aims to provide a mutant with thermal stability, a primer pair for amplifying the nucleic acid sequence of the mutant site of the L-lysine oxidase mutant, and a soluble protein, an enzyme preparation, a recombinant cell and a recombinant vector containing the mutant, which are used for catalyzing and oxidizing L-lysine, aiming at the problem of insufficient thermal stability of the existing L-lysine oxidase mutant.

According to a first aspect of the present disclosure, there is provided a mutant having an amino acid sequence of (a) or (b) as follows:

(a) at least one amino acid of the amino acid sequence SEQ ID NO.1 of the natural L-lysine oxidase is substituted, deleted or added, and the amino acid sequence of the mutant has more than 90 percent of homology with the amino acid sequence SEQ ID NO. 1; or

(b) At least one amino acid of the amino acid sequence SEQ ID NO.1 of the natural L-lysine oxidase is substituted, deleted or added, and the amino acid sequence of the mutant has the same function as that of the amino acid sequence SEQ ID NO. 1.

In some possible implementations, the amino acid sequence of the mutant is configured to include the amino acid sequence of one or more of the mutation sites T122V, D229N, a417V, G438D on SEQ ID No. 1.

In some possible implementations, the amino acid sequence of the mutant is any one of SEQ ID No. 2-16.

According to a second aspect of the present disclosure, there is provided a gene sequence encoding the aforementioned mutant, which is configured as any one of SEQ ID No.17 to 31, wherein:

the nucleic acid sequence of the mutant with the mutation site of T122V is SEQ ID NO. 17;

the nucleic acid sequence of the mutant with the mutation site of D229N is SEQ ID NO. 18;

the nucleic acid sequence of the mutant with the mutation site A417V is SEQ ID NO. 19;

the nucleic acid sequence of the mutant with the mutation site of G438D is SEQ ID NO. 20;

the nucleic acid sequence encoding the mutant with mutation sites T122V and D229N is SEQ ID No. 21;

the nucleic acid sequence encoding the mutant with the mutation sites of T122V and A417V is SEQ ID NO. 22;

the nucleic acid sequence of the mutant with the mutation sites of T122V and G438D is SEQ ID NO. 23;

the nucleic acid sequence encoding the mutant with the mutation sites of D229N and A417V is SEQ ID NO. 24;

the nucleic acid sequence of the mutant with the mutation sites of D229N and G438D is SEQ ID NO. 25;

the nucleic acid sequence of the mutant with the mutation sites of A417V and G438D is SEQ ID NO. 26;

the nucleic acid sequence of the L-lysine oxidase mutant encoding the mutants with mutation sites of T122V, D229N and A417V is SEQ ID NO. 27;

the nucleic acid sequence of the L-lysine oxidase mutant encoding the mutant with mutation sites of T122V, D229N and G438D is SEQ ID NO. 28;

the nucleic acid sequence of the mutant which codes for the mutation sites D229N, A417V and G438D is SEQ ID NO. 29;

the nucleic acid sequence of the mutant with the mutation sites of T122V, A417V and G438D is SEQ ID NO. 30;

the nucleic acid sequence encoding the mutants with mutation sites T122V, D229N, A417V and G438D is SEQ ID NO. 31.

Further, the nucleic acid sequences of the amplification primer pair of the mutation site T122V are SEQ ID NO.32 and SEQ ID NO. 33.

Further, the nucleic acid sequences of the amplification primer pair of the mutation site D229N are SEQ ID NO.34 and SEQ ID NO. 35.

Further, the nucleic acid sequences of the amplification primer pair of the mutation site A417V are SEQ ID NO.36 and SEQ ID NO. 37.

Further, the nucleic acid sequences of the amplification primer pair of the mutation site G438D are SEQ ID NO.38 and SEQ ID NO. 39.

According to a third aspect of the present disclosure, there is provided a method for constructing the aforementioned mutant, the method comprising:

obtaining an amino acid sequence SEQ ID NO.1 of natural L-lysine oxidase in a database;

removing the repetitive sequence and the redundant sequence in the SEQ ID NO.1, and selecting an amino acid sequence with the consistency of more than 50 percent with the SEQ ID NO. 1;

carrying out multi-sequence comparison on an amino acid sequence with more than 50% of consistency with SEQ ID NO.1, and then carrying out protein three-dimensional structure prediction on the compared amino acid sequence;

and screening out mutation sites related to thermal stability.

According to a fourth aspect of the present disclosure, there is provided a soluble protein configured to comprise a mutant as described above.

According to a fifth aspect of the present disclosure, there is provided an immobilized enzyme configured to comprise a mutant as described above.

According to a sixth aspect of the present disclosure, there is provided a recombinant cell configured to include a coding sequence of a mutant as described above.

According to a seventh aspect of the present disclosure, there is provided a recombinant vector configured to include a coding sequence of a mutant as described above.

According to an eighth aspect of the present disclosure, there is provided the use of the aforementioned mutant, soluble protein, enzyme preparation, recombinant cell or recombinant vector for the catalytic oxidation of L-lysine.

The L-lysine oxidase LysOX mutant with improved thermal stability provided by the invention comprises a single-point mutant and a combined mutant, and compared with the wild type L-lysine oxidase LysOX, the single-point mutant and the combined mutant have longer half-lives at 45 ℃; in particular, the combination mutant showed a synergistic effect of thermal stability of the single-point mutant, with a half-life of about 3 times that of the wild-type L-lysOX. Based on the above, the L-lysine oxidase LysOX mutant provided by the invention has better thermal stability, and is suitable for catalyzing and oxidizing L-lysine at higher temperature.

The construction method of the L-lysine oxidase LysOX mutant with improved thermal stability is different from the rational design based on the precise structure-function relationship of protein, the construction method takes the Consensus Concept as a guide idea, analyzes information capable of improving the thermal stability of the enzyme from the aspect of evolution, performs integration analysis on L-amino acid oxidase family sequences, and combines the assistance of bioinformatics and crystallography methods to obtain the novel L-lysine oxidase LysOX mutant with high stability.

The L-lysine oxidase LysOX mutant with improved thermal stability provided by the invention has a better application prospect in the aspect of L-lysine detection.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

Fig. 1 shows a schematic diagram of a simulated crystal structure of L-lys oxidase LysOX protein and a schematic diagram of distribution of mutation sites on the crystal structure according to an embodiment of the present disclosure.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

The present disclosure provides an L-lys oxidase LysOX mutant with improved thermal stability, wherein the L-lys oxidase LysOX is a wild-type L-lys oxidase LysOX derived from Trichoderma viride, and is named as LysOX protein, and the amino acid sequence of the LysOX protein is SEQ ID No. 1.

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

(a) at least one amino acid of the amino acid sequence SEQ ID NO.1 of the natural L-lysine oxidase is substituted, deleted or added, and the amino acid sequence of the mutant has more than 90 percent of homology with the amino acid sequence SEQ ID NO. 1; or

(b) At least one amino acid of the amino acid sequence SEQ ID NO.1 of the natural L-lysine oxidase is substituted, deleted or added, and the amino acid sequence of the mutant has the same function as that of the amino acid sequence SEQ ID NO. 1.

In some embodiments, the amino acid sequence of the mutant is any one of SEQ ID NO. 2-16. Specifically, a certain site is selected from the amino acid sequence shown in SEQ ID No.1 for single-point mutation, and 5L-lysine oxidase single-point mutants are obtained respectively, wherein the mutation sites are as follows: S95A, T122V, D229N, A417V and G438D, and the 5L-lysine oxidase single-point mutants are subjected to property measurement to screen 4L-lysine oxidase mutants with improved thermal stability, wherein the mutation sites of the L-lysine oxidase mutants are as follows: T122V, D229N, A417V, G438D, the amino acid sequences of which are SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and,

selecting a plurality of mutation sites for combination in the amino acid sequence shown in SEQ ID NO. 1:

1) for example, 2 mutation sites are selected from the 4 mutation sites to be combined to obtain T122V/D229N, T122V/A417V, T122V/G438D, D229N/A417V, D229N/G438D and A417V/G438D, and the amino acid sequences are respectively 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.

2) For example, 3 mutation sites are selected from the 4 mutation sites and combined to obtain 4L-lysine oxidase mutants with improved thermal stability, wherein the combined mutation sites are as follows:

T122V/D229N/A417V，

T122V/D229N/G438D，

D229N/A417V/G438D，

T122V/A417V/G438D, and the amino acid sequences are SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15 respectively.

3) For example, 4 mutation sites are selected from the 4 mutation sites and combined to obtain 1L-lysine oxidase mutant with improved heat stability, wherein the combined mutation sites are as follows:

T122V/D229N/A417V/G438D, the amino acid sequence of which is SEQ ID NO. 16.

The embodiments of the present disclosure provide a gene sequence encoding the aforementioned mutant, which corresponds to the amino acid sequences having different mutation sites as follows:

the nucleic acid sequence of the mutant with the mutation site of T122V is SEQ ID NO. 17;

the nucleic acid sequence of the mutant with the mutation site of D229N is SEQ ID NO. 18;

the nucleic acid sequence of the mutant with the mutation site A417V is SEQ ID NO. 19;

the nucleic acid sequence of the mutant with the mutation site of G438D is SEQ ID NO. 20;

the nucleic acid sequence encoding the mutant with mutation sites T122V and D229N is SEQ ID No. 21;

the nucleic acid sequence encoding the mutant with the mutation sites of T122V and A417V is SEQ ID NO. 22;

the nucleic acid sequence of the mutant with the mutation sites of T122V and G438D is SEQ ID NO. 23;

the nucleic acid sequence encoding the mutant with the mutation sites of D229N and A417V is SEQ ID NO. 24;

the nucleic acid sequence of the mutant with the mutation sites of D229N and G438D is SEQ ID NO. 25;

the nucleic acid sequence of the mutant with the mutation sites of A417V and G438D is SEQ ID NO. 26;

the nucleic acid sequence of the L-lysine oxidase mutant encoding the mutants with mutation sites of T122V, D229N and A417V is SEQ ID NO. 27;

the nucleic acid sequence of the L-lysine oxidase mutant encoding the mutant with mutation sites of T122V, D229N and G438D is SEQ ID NO. 28;

the nucleic acid sequence of the mutant which codes for the mutation sites D229N, A417V and G438D is SEQ ID NO. 29;

the nucleic acid sequence of the mutant with the mutation sites of T122V, A417V and G438D is SEQ ID NO. 30;

the nucleic acid sequence encoding the mutants with mutation sites T122V, D229N, A417V and G438D is SEQ ID NO. 31.

Further, the nucleic acid sequences of the amplification primer pair of the mutation site T122V are SEQ ID NO.32 and SEQ ID NO. 33.

Further, the nucleic acid sequences of the amplification primer pair of the mutation site D229N are SEQ ID NO.34 and SEQ ID NO. 35.

Further, the nucleic acid sequences of the amplification primer pair of the mutation site A417V are SEQ ID NO.36 and SEQ ID NO. 37.

Further, the nucleic acid sequences of the amplification primer pair of the mutation site G438D are SEQ ID NO.38 and SEQ ID NO. 39.

The embodiment of the disclosure also provides a method for constructing the mutant, which comprises the following steps:

cloning of the S1 wild-type L-lysine oxidase LysOX Gene

And (3) carrying out codon optimization on the wild L-lysine oxidase gene by taking escherichia coli as a host cell to obtain an optimized LysOX gene, wherein the expressed amino acid sequence of the optimized LysOX gene is SEQ ID NO. 1.

Using SEQ ID NO.1 as a target gene, and adopting the following amplification primer pairs to amplify the target gene:

F：5’-CGCATATGATGGACAACGTAGACTTCGCAGAGTCAG-3' (wherein the restriction enzyme NdeI recognition site is underlined);

R：5’-TCAGCTCTCGAGCTTTACCTGGTACTCCTTTGGTAG-3' (wherein the restriction enzyme XhoI recognition site is underlined).

The amplification conditions were: amplification was carried out at 95 ℃ for 2min, followed by amplification at 56 ℃ for 20sec, at 72 ℃ for 90sec for 30 cycles, and finally at 72 ℃ for 10 min.

After the reaction is finished, detecting the PCR amplification product by 1.5% agarose gel electrophoresis to obtain a 1.0kb band, wherein the length of the band accords with an expected result. The desired fragment was recovered and purified by the standard procedures of a kit, and the desired fragment and pET28a plasmid were digested simultaneously with restriction endonucleases XhoI and NdeI, and then ligated with T4 DNA ligase, and the resulting ligation product was transformed into E.coli BL21(DE3) competent cells, and the transformed cells were plated on LB plate containing 100. mu.g/ml kanamycin to extract positive clone plasmid and sequenced, as a result, it was revealed that the cloned L-lysine oxidase LysOX gene had the correct sequence and that pET28a plasmid had been ligated correctly, and recombinant plasmid pET28a-LysOX was obtained.

Wherein, the PCR amplification enzyme is KOD high fidelity polymerase.

Expression and purification of S2 LysOX protein

The recombinant plasmid pET28a-LysOX in the glycerin pipe is inoculated into a 4mL LB culture medium test tube containing 100 mu g/mL Kan according to the volume ratio of 1 percent, and cultured for 12h at 37 ℃ and 220 rpm; transferring 4mL of the bacterial liquid into a shake flask containing 1L LB culture medium of 100 mu g/mL Kan, culturing at 37 ℃ and 220rpm for 2.5h to make OD600 reach 0.6-0.8, adding 1mM IPTG inducer, and performing induction culture at 25 ℃ and 200rpm for 14 h. And ultrasonically crushing the escherichia coli thallus suspension obtained after fermentation, and performing Ni-NTA affinity chromatography treatment to obtain the LysOX protein with the purity of more than 95%, wherein the amino acid sequence is SEQ ID NO. 1.

Multiple sequence alignment and Consensus analysis of S3 LysOX homologous proteins

S301: entering a Pfam database homepage (http:// Pfam. xfam. org /), inputting an amino acid SEQUENCE of LysOX in a SEQUENCE SEARCH tool for searching, directly feeding back an alignment result of the amino acid SEQUENCE of the whole family of the protein by a server, displaying the abundance of various amino acids of each mutation site in a bar graph mode, and automatically generating a consensus SEQUENCE of the protein family by the website;

s302: inputting an amino acid sequence shown by SEQ ID NO.2 into an NCBI protein database and a Pfam database, finding out all protein sequences with the consistency of more than 50 percent with an amino acid sequence (SEQ ID NO.2) of the LysOX protein by using a Blast tool, deleting the repeated identical sequences in the protein sequences, arranging the rest amino acid sequences into a fasta format, inputting Clustalx1.83 software for multi-sequence comparison, and outputting comparison results in an aln, dnd and fasta format, wherein the dnd file is used for constructing an evolutionary tree file, and the aln and fasta files are sequence files with different forms;

uploading the fasta file to Consensus Maker v2.0.0

(https://www.hiv.lanl.gov/content/sequence/CONSENSUS/consensus.htm l)

And after the set parameters are modified according to the needs, the online software generates a consensus sequence which can be edited in a later period.

S303: the amino acid sequence of LysOX protein was compared against the family consensus sequence and the amino acid abundance map at each site.

S4: simulation of three-dimensional structure of LysOX protein and selection of mutation hot spots

S401: obtaining the prediction of the three-dimensional structure of the LysOX protein (amino acid sequence SEQ ID NO.1) by a swisscodel online tool;

s402: the crystal structure of the LysOX protein (amino acid sequence SEQ ID NO.1) is observed by PyMOL, the mutant site and the mutant form to be selected are reviewed according to the structural information, and the mutant site which is most likely to improve the thermal stability of the LysOX protein is selected, wherein the selection conditions are as follows:

(1) the standard for judging a certain locus as a candidate locus is as follows:

the amino acid abundance of most proteins in the family at the position is high overall;

② the amino acid at the site is conserved;

and the amino acid with higher occurrence frequency at the site has larger physical and chemical property difference with the amino acid of the LysOX protein at the site, such as hydrogen bonds, charge difference, polarity strength, steric hindrance and the like.

(2) Removal of the active site in the vicinity, i.e.away from the catalytic residue (glutamic acid position 104)

Amino acid residues within the range, excluding amino acid residues in the embedded or semi-embedded state.

After the above two-step screening, there remain 21 different sites, most of which are located on the surface of the LysOX protein molecule, as shown in fig. 1, and the arrows indicate the mutation sites.

(3) The above 21 mutant forms were analyzed in detail one by one based on the crystal structure of LysOX protein, and mutants that could improve the thermostability of LysOX protein were selected.

The main judgment criteria are: firstly, the mutation eliminates the original acting force form which is not beneficial to thermal stability, such as electrostatic repulsion, charge aggregation and the like; secondly, the mutation does not damage the existing acting force form which is beneficial to thermal stability and the stable protein structure; and thirdly, new acting force forms which are beneficial to thermal stability, such as hydrogen bonds, salt bridges, hydrophobic interaction and the like, are introduced into the mutation.

Totally designing 5 single-point mutants, wherein the mutation sites are respectively as follows:

S95A、T122V、D229N、A417V、G438D；

and (3) carrying out activity determination on the 5L-lysine oxidase single-point mutants to screen out 4L-lysine oxidase mutants with improved thermal stability, wherein the mutation sites are as follows: the amino acid sequences of the corresponding single-point mutants of T122V, D229N, A417V and G438D are SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 respectively.

Construction, expression and purification of S5 mutant

S501: construction of LysOX protein Single Point mutants

Carrying out whole plasmid PCR amplification by using KOD high fidelity enzyme by using a recombinant plasmid pET28a-LysOX in S1 as a template and a pair of complementary oligonucleotides with mutation sites as amplification primers to obtain a recombinant plasmid with a specific mutation site;

the amplification primer pairs used were:

(1) the nucleic acid sequences of the amplification primers upstream and downstream of the mutation site T122V are as follows:

F(SEQ ID NO.32)：

5'-GAGTCATCATCACGAACTGGAGGACGACTATACACACAC-3'；

R(SEQ ID NO.33)：

5'-GTGTGTGTATAGTCGTCCTCCAGTTCGTGATGATGACTC-3'；

(2) the nucleic acid sequences of the amplification primers upstream and downstream of the mutation site D229N were as follows:

F(SEQ ID NO.34)：

5'-GAGAAGCTAGCAGAGAACTTCGACAAGGGATTCGACGA-3'；

R(SEQ ID NO.35)：

5'-TCGTCGAATCCCTTGTCGAAGTTCTCTGCTAGCTTCTC-3'；

(3) the nucleic acid sequences of the amplification primers upstream and downstream of mutation site a417V are as follows:

F(SEQ ID NO.36)：

5'-AATTACATGCGGAGGAGTAGCATCAACAGACCTACCAC-3'；

R(SEQ ID NO.37)：

5'-GTGGTAGGTCTGTTGATGCTACTCCTCCGCATGTAATT-3'；

(4) the nucleic acid sequences of the upstream and downstream amplification primers at mutation site G438D are as follows:

F(SEQ ID NO.38)：

5'-AACCTAGGAGACACAGACGAGGCAGTACTACTAGCATC-3'；

R(SEQ ID NO.39)：

5'-GATGCTAGTAGTACTGCCTCGTCTGTGTCTCCTAGGTT-3'；

the amplification conditions were: amplifying at 95 ℃ for 2min, then at 56 ℃ for 20sec, at 72 ℃ for 90sec for 30 cycles, and finally at 72 ℃ for 10 min; recovering PCR amplification products by glue, digesting the glue recovery products for 2h at 37 ℃ by using DpnI enzyme, and degrading the initial template; and (3) transforming the digestion product into escherichia coli BL21(DE3) competent cells, coating the cells on an LB agar plate containing 100 mu g/mL kanamycin, carrying out overnight culture at 37 ℃, screening positive clones, and carrying out sequencing verification to obtain the recombinant bacteria containing the L-lysine oxidase single-point mutant.

S502: construction of LysOX protein combination mutants

Using a construction method similar to the single-point mutant to cumulatively combine the single-point mutants with improved stability, selecting a plurality of mutation sites for combination in the amino acid sequence shown in SEQ ID No.1, for example, selecting 2-4 mutation sites from the 4 mutation sites for combination to respectively obtain different L-lysine oxidase combined mutants:

(1) 2 mutation sites are selected for combination, 6L-lysyl oxidase mutants with improved thermal stability and L-lysyl oxidase combined mutants can be constructed, and the combined mutation sites are respectively as follows:

T122V/D229N、T122V/A417V、T122V/G438D、D229N/A417V、D229N/G438D、A417V/G438D，

the amino acid sequences of the 6L-lysine oxidase combined mutants with improved thermal stability are respectively 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;

(2) selecting 3 mutation sites for combination, 4L-lysine oxidase combined mutants with improved thermal stability can be constructed, wherein the combined mutation sites are respectively as follows:

T122V/D229N/A417V、T122V/D229N/G438D、D229N/A417V/G438D、T122V/A417V/G438D，

the amino acid sequences of the 4L-lysine oxidase combined mutants with improved thermal stability are respectively SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO. 15;

(3) 4 mutation sites are selected for combination, 1L-lysine oxidase combined mutant with improved heat stability can be constructed, and the combined mutation sites are respectively as follows:

T122V/D229N/A417V/G438D，

the amino acid sequence of the 1L-lysine oxidase combination mutant with improved thermal stability is SEQ ID NO. 16.

Experiment-characterization of enzymatic Properties of L-lysine oxidase mutants

The natural wild type L-lysine oxidase and the various L-lysine oxidase mutants provided by the embodiment are subjected to a thermal stability test, and according to a conventional L-lysine oxidase activity determination method, the method specifically comprises the following steps:

incubating the enzyme solution at a certain temperature, sampling at different treatment times, determining the percent residual activity of the L-lysine oxidase or L-lysine oxidase mutant, and plotting the ln value of the percent residual activity against the time t (min), wherein the slope of the straight line is the inactivation constant k_inactFrom t1/2 ═ ln2/k_inactObtaining the half-life period of the wild L-lysine oxidase or the mutant L-lysine oxidase at the temperature.

The experimental results show that the thermal stability of 4 single-point mutants and 11 combination mutants in the above L-lysine oxidase mutants is obviously improved, as shown in Table 1:

TABLE 1 characterization of enzymatic Properties of wild-type L-lysine oxidase, Single-site mutants and combination mutants

From table 1, it can be seen that the L-lysyl oxidase mutants provided by the present invention include single-site mutants and combination mutants, which have longer half-lives at 45 ℃ than wild-type L-lysyl oxidase; especially the combination mutant, shows the additive effect of the thermal stability of the single-point mutant, and the half-life period of the combination mutant is about 3 times of that of the wild-type L-lysine oxidase. Based on the above, the L-lysine oxidase mutant provided by the invention has better thermal stability, and is suitable for catalyzing and oxidizing L-lysine at higher temperature.

While the invention has been illustrated and described in further detail by preferred embodiments, the invention is not limited to the disclosed examples and other variations can be derived therefrom by those skilled in the art without departing from the scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

Sequence listing

<110> institute of biomedical engineering technology of Suzhou, China academy of sciences

<160>39

<210>1

<211>617

<212>PRT

<213> Artificial

<400>1

MDNVDFAESV RTRWARRLIR EKVAKELNIL TERLGEVPGI PPPREGRFLG GGYSHDNLPS 60

DPLYSSIKPA LLKEAPRAEE ELPPRKVCIV GAGVSGLYIA MILDDLKIPN LTYDIFESSS 120

RTGGRLYTHH FTDAKHDYYD IGAMRYPDIP SMKRTFNLFK RTGMPLIKYY LDGENTPQLY 180

NNHFFAKGVV DPYMVSVANG GTVPDDVVDS VGEKLQQAFG YYKEKLAEDF DKGFDELMLV 240

DDMTTREYLK RGGPKGEAPK YDFFAIQWME TQNTGTNLFD QAFSESVIDS FDFDNPTKPE 300

WYCIEGGTSL LVDAMKETLV HKVQNNKRVE AISIDLDAPD DGNMSVKIGG KDYSGYSTVF 360

NTTALGCLDR MDLRGLNLHP TQADAIRCLH YDNSTKVALK FSYPWWIKDC GITCGGAAST 420

DLPLRTCVYP SYNLGDTGEA VLLASYTWSQ DATRIGSLVK DAPPQPPKED ELVELILQNL 480

ARLHAEHMTY EKIKEAYTGV YHAYCWANDP NVGGAFALFG PGQFSNLYPY LMRPAAGGKF 540

HIVGEASSVH HAWIIGSLES AYTAVYQFLY KYKMWDYLRL LLERWQYGLQ ELETGKHGTA 600

HLQFILGSLP KEYQVKI 617

<210>2

<211>617

<212> PRT

<400>2

MDNVDFAESV RTRWARRLIR EKVAKELNIL TERLGEVPGI PPPREGRFLG GGYSHDNLPS 60

DPLYSSIKPA LLKEAPRAEE ELPPRKVCIV GAGVSGLYIA MILDDLKIPN LTYDIFESSS 120

RVGGRLYTHH FTDAKHDYYD IGAMRYPDIP SMKRTFNLFK RTGMPLIKYY LDGENTPQLY 180

NNHFFAKGVV DPYMVSVANG GTVPDDVVDS VGEKLQQAFG YYKEKLAEDF DKGFDELMLV 240

DDMTTREYLK RGGPKGEAPK YDFFAIQWME TQNTGTNLFD QAFSESVIDS FDFDNPTKPE 300

WYCIEGGTSL LVDAMKETLV HKVQNNKRVE AISIDLDAPD DGNMSVKIGG KDYSGYSTVF 360

NTTALGCLDR MDLRGLNLHP TQADAIRCLH YDNSTKVALK FSYPWWIKDC GITCGGAAST 420

DLPLRTCVYP SYNLGDTGEA VLLASYTWSQ DATRIGSLVK DAPPQPPKED ELVELILQNL 480

ARLHAEHMTY EKIKEAYTGV YHAYCWANDP NVGGAFALFG PGQFSNLYPY LMRPAAGGKF 540

HIVGEASSVH HAWIIGSLES AYTAVYQFLY KYKMWDYLRL LLERWQYGLQ ELETGKHGTA 600

HLQFILGSLP KEYQVKI 617

<210>3

<211>617

<212>PRT

<213> Artificial

<400>3

MDNVDFAESV RTRWARRLIR EKVAKELNIL TERLGEVPGI PPPREGRFLG GGYSHDNLPS 60

DPLYSSIKPA LLKEAPRAEE ELPPRKVCIV GAGVSGLYIA MILDDLKIPN LTYDIFESSS 120

RTGGRLYTHH FTDAKHDYYD IGAMRYPDIP SMKRTFNLFK RTGMPLIKYY LDGENTPQLY 180

NNHFFAKGVV DPYMVSVANG GTVPDDVVDS VGEKLQQAFG YYKEKLAENF DKGFDELMLV 240

DDMTTREYLK RGGPKGEAPK YDFFAIQWME TQNTGTNLFD QAFSESVIDS FDFDNPTKPE 300

WYCIEGGTSL LVDAMKETLV HKVQNNKRVE AISIDLDAPD DGNMSVKIGG KDYSGYSTVF 360

NTTALGCLDR MDLRGLNLHP TQADAIRCLH YDNSTKVALK FSYPWWIKDC GITCGGAAST 420

DLPLRTCVYP SYNLGDTGEA VLLASYTWSQ DATRIGSLVK DAPPQPPKED ELVELILQNL 480

ARLHAEHMTY EKIKEAYTGV YHAYCWANDP NVGGAFALFG PGQFSNLYPY LMRPAAGGKF 540

HIVGEASSVH HAWIIGSLES AYTAVYQFLY KYKMWDYLRL LLERWQYGLQ ELETGKHGTA 600

HLQFILGSLP KEYQVKI 617

<210>4

<211>617

<212>PRT

<213> Artificial

<400>4

MDNVDFAESV RTRWARRLIR EKVAKELNIL TERLGEVPGI PPPREGRFLG GGYSHDNLPS 60

DPLYSSIKPA LLKEAPRAEE ELPPRKVCIV GAGVSGLYIA MILDDLKIPN LTYDIFESSS 120

RTGGRLYTHH FTDAKHDYYD IGAMRYPDIP SMKRTFNLFK RTGMPLIKYY LDGENTPQLY 180

NNHFFAKGVV DPYMVSVANG GTVPDDVVDS VGEKLQQAFG YYKEKLAEDF DKGFDELMLV 240

DDMTTREYLK RGGPKGEAPK YDFFAIQWME TQNTGTNLFD QAFSESVIDS FDFDNPTKPE 300

WYCIEGGTSL LVDAMKETLV HKVQNNKRVE AISIDLDAPD DGNMSVKIGG KDYSGYSTVF 360

NTTALGCLDR MDLRGLNLHP TQADAIRCLH YDNSTKVALK FSYPWWIKDC GITCGGVAST 420

DLPLRTCVYP SYNLGDTGEA VLLASYTWSQ DATRIGSLVK DAPPQPPKED ELVELILQNL 480

ARLHAEHMTY EKIKEAYTGV YHAYCWANDP NVGGAFALFG PGQFSNLYPY LMRPAAGGKF 540

HIVGEASSVH HAWIIGSLES AYTAVYQFLY KYKMWDYLRL LLERWQYGLQ ELETGKHGTA 600

HLQFILGSLP KEYQVKI 617

<210>5

<211>617

<212>PRT

<213> Artificial

<400>5

MDNVDFAESV RTRWARRLIR EKVAKELNIL TERLGEVPGI PPPREGRFLG GGYSHDNLPS 60

DPLYSSIKPA LLKEAPRAEE ELPPRKVCIV GAGVSGLYIA MILDDLKIPN LTYDIFESSS 120

RTGGRLYTHH FTDAKHDYYD IGAMRYPDIP SMKRTFNLFK RTGMPLIKYY LDGENTPQLY 180

NNHFFAKGVV DPYMVSVANG GTVPDDVVDS VGEKLQQAFG YYKEKLAEDF DKGFDELMLV 240

DDMTTREYLK RGGPKGEAPK YDFFAIQWME TQNTGTNLFD QAFSESVIDS FDFDNPTKPE 300

WYCIEGGTSL LVDAMKETLV HKVQNNKRVE AISIDLDAPD DGNMSVKIGG KDYSGYSTVF 360

NTTALGCLDR MDLRGLNLHP TQADAIRCLH YDNSTKVALK FSYPWWIKDC GITCGGAAST 420

DLPLRTCVYP SYNLGDTDEA VLLASYTWSQ DATRIGSLVK DAPPQPPKED ELVELILQNL 480

ARLHAEHMTY EKIKEAYTGV YHAYCWANDP NVGGAFALFG PGQFSNLYPY LMRPAAGGKF 540

HIVGEASSVH HAWIIGSLES AYTAVYQFLY KYKMWDYLRL LLERWQYGLQ ELETGKHGTA 600

HLQFILGSLP KEYQVKI 617

<210>6

<211>617

<212>PRT

<213> Artificial

<400>6

MDNVDFAESV RTRWARRLIR EKVAKELNIL TERLGEVPGI PPPREGRFLG GGYSHDNLPS 60

DPLYSSIKPA LLKEAPRAEE ELPPRKVCIV GAGVSGLYIA MILDDLKIPN LTYDIFESSS 120

RVGGRLYTHH FTDAKHDYYD IGAMRYPDIP SMKRTFNLFK RTGMPLIKYY LDGENTPQLY 180

NNHFFAKGVV DPYMVSVANG GTVPDDVVDS VGEKLQQAFG YYKEKLAENF DKGFDELMLV 240

DDMTTREYLK RGGPKGEAPK YDFFAIQWME TQNTGTNLFD QAFSESVIDS FDFDNPTKPE 300

WYCIEGGTSL LVDAMKETLV HKVQNNKRVE AISIDLDAPD DGNMSVKIGG KDYSGYSTVF 360

NTTALGCLDR MDLRGLNLHP TQADAIRCLH YDNSTKVALK FSYPWWIKDC GITCGGAAST 420

DLPLRTCVYP SYNLGDTGEA VLLASYTWSQ DATRIGSLVK DAPPQPPKED ELVELILQNL 480

ARLHAEHMTY EKIKEAYTGV YHAYCWANDP NVGGAFALFG PGQFSNLYPY LMRPAAGGKF 540

HIVGEASSVH HAWIIGSLES AYTAVYQFLY KYKMWDYLRL LLERWQYGLQ ELETGKHGTA 600

HLQFILGSLP KEYQVKI 617

<210>7

<211>617

<212>PRT

<213> Artificial

<400>7

MDNVDFAESV RTRWARRLIR EKVAKELNIL TERLGEVPGI PPPREGRFLG GGYSHDNLPS 60

DPLYSSIKPA LLKEAPRAEE ELPPRKVCIV GAGVSGLYIA MILDDLKIPN LTYDIFESSS 120

RVGGRLYTHH FTDAKHDYYD IGAMRYPDIP SMKRTFNLFK RTGMPLIKYY LDGENTPQLY 180

NNHFFAKGVV DPYMVSVANG GTVPDDVVDS VGEKLQQAFG YYKEKLAEDF DKGFDELMLV 240

DDMTTREYLK RGGPKGEAPK YDFFAIQWME TQNTGTNLFD QAFSESVIDS FDFDNPTKPE 300

WYCIEGGTSL LVDAMKETLV HKVQNNKRVE AISIDLDAPD DGNMSVKIGG KDYSGYSTVF 360

NTTALGCLDR MDLRGLNLHP TQADAIRCLH YDNSTKVALK FSYPWWIKDC GITCGGVAST 420

DLPLRTCVYP SYNLGDTGEA VLLASYTWSQ DATRIGSLVK DAPPQPPKED ELVELILQNL 480

ARLHAEHMTY EKIKEAYTGV YHAYCWANDP NVGGAFALFG PGQFSNLYPY LMRPAAGGKF 540

HIVGEASSVH HAWIIGSLES AYTAVYQFLY KYKMWDYLRL LLERWQYGLQ ELETGKHGTA 600

HLQFILGSLP KEYQVKI 617

<210>8

<211>617

<212>PRT

<213> Artificial

<400>8

MDNVDFAESV RTRWARRLIR EKVAKELNIL TERLGEVPGI PPPREGRFLG GGYSHDNLPS 60

DPLYSSIKPA LLKEAPRAEE ELPPRKVCIV GAGVSGLYIA MILDDLKIPN LTYDIFESSS 120

RVGGRLYTHH FTDAKHDYYD IGAMRYPDIP SMKRTFNLFK RTGMPLIKYY LDGENTPQLY 180

NNHFFAKGVV DPYMVSVANG GTVPDDVVDS VGEKLQQAFG YYKEKLAEDF DKGFDELMLV 240

DDMTTREYLK RGGPKGEAPK YDFFAIQWME TQNTGTNLFD QAFSESVIDS FDFDNPTKPE 300

WYCIEGGTSL LVDAMKETLV HKVQNNKRVE AISIDLDAPD DGNMSVKIGG KDYSGYSTVF 360

NTTALGCLDR MDLRGLNLHP TQADAIRCLH YDNSTKVALK FSYPWWIKDC GITCGGAAST 420

DLPLRTCVYP SYNLGDTDEA VLLASYTWSQ DATRIGSLVK DAPPQPPKED ELVELILQNL 480

ARLHAEHMTY EKIKEAYTGV YHAYCWANDP NVGGAFALFG PGQFSNLYPY LMRPAAGGKF 540

HIVGEASSVH HAWIIGSLES AYTAVYQFLY KYKMWDYLRL LLERWQYGLQ ELETGKHGTA 600

HLQFILGSLP KEYQVKI 617

<210>9

<211>617

<212>PRT

<213> Artificial

<400>9

MDNVDFAESV RTRWARRLIR EKVAKELNIL TERLGEVPGI PPPREGRFLG GGYSHDNLPS 60

DPLYSSIKPA LLKEAPRAEE ELPPRKVCIV GAGVSGLYIA MILDDLKIPN LTYDIFESSS 120

RTGGRLYTHH FTDAKHDYYD IGAMRYPDIP SMKRTFNLFK RTGMPLIKYY LDGENTPQLY 180

NNHFFAKGVV DPYMVSVANG GTVPDDVVDS VGEKLQQAFG YYKEKLAENF DKGFDELMLV 240

DDMTTREYLK RGGPKGEAPK YDFFAIQWME TQNTGTNLFD QAFSESVIDS FDFDNPTKPE 300

WYCIEGGTSL LVDAMKETLV HKVQNNKRVE AISIDLDAPD DGNMSVKIGG KDYSGYSTVF 360

NTTALGCLDR MDLRGLNLHP TQADAIRCLH YDNSTKVALK FSYPWWIKDC GITCGGVAST 420

DLPLRTCVYP SYNLGDTGEA VLLASYTWSQ DATRIGSLVK DAPPQPPKED ELVELILQNL 480

ARLHAEHMTY EKIKEAYTGV YHAYCWANDP NVGGAFALFG PGQFSNLYPY LMRPAAGGKF 540

HIVGEASSVH HAWIIGSLES AYTAVYQFLY KYKMWDYLRL LLERWQYGLQ ELETGKHGTA 600

HLQFILGSLP KEYQVKI 617

<210>10

<211>617

<212>PRT

<213> Artificial

<400>10

MDNVDFAESV RTRWARRLIR EKVAKELNIL TERLGEVPGI PPPREGRFLG GGYSHDNLPS 60

DPLYSSIKPA LLKEAPRAEE ELPPRKVCIV GAGVSGLYIA MILDDLKIPN LTYDIFESSS 120

RTGGRLYTHH FTDAKHDYYD IGAMRYPDIP SMKRTFNLFK RTGMPLIKYY LDGENTPQLY 180

NNHFFAKGVV DPYMVSVANG GTVPDDVVDS VGEKLQQAFG YYKEKLAENF DKGFDELMLV 240

DDMTTREYLK RGGPKGEAPK YDFFAIQWME TQNTGTNLFD QAFSESVIDS FDFDNPTKPE 300

WYCIEGGTSL LVDAMKETLV HKVQNNKRVE AISIDLDAPD DGNMSVKIGG KDYSGYSTVF 360

NTTALGCLDR MDLRGLNLHP TQADAIRCLH YDNSTKVALK FSYPWWIKDC GITCGGAAST 420

DLPLRTCVYP SYNLGDTDEA VLLASYTWSQ DATRIGSLVK DAPPQPPKED ELVELILQNL 480

ARLHAEHMTY EKIKEAYTGV YHAYCWANDP NVGGAFALFG PGQFSNLYPY LMRPAAGGKF 540

HIVGEASSVH HAWIIGSLES AYTAVYQFLY KYKMWDYLRL LLERWQYGLQ ELETGKHGTA 600

HLQFILGSLP KEYQVKI 617

<210>11

<211>617

<212>PRT

<213> Artificial

<400>11

MDNVDFAESV RTRWARRLIR EKVAKELNIL TERLGEVPGI PPPREGRFLG GGYSHDNLPS 60

DPLYSSIKPA LLKEAPRAEE ELPPRKVCIV GAGVSGLYIA MILDDLKIPN LTYDIFESSS 120

RTGGRLYTHH FTDAKHDYYD IGAMRYPDIP SMKRTFNLFK RTGMPLIKYY LDGENTPQLY 180

NNHFFAKGVV DPYMVSVANG GTVPDDVVDS VGEKLQQAFG YYKEKLAEDF DKGFDELMLV 240

DDMTTREYLK RGGPKGEAPK YDFFAIQWME TQNTGTNLFD QAFSESVIDS FDFDNPTKPE 300

WYCIEGGTSL LVDAMKETLV HKVQNNKRVE AISIDLDAPD DGNMSVKIGG KDYSGYSTVF 360

NTTALGCLDR MDLRGLNLHP TQADAIRCLH YDNSTKVALK FSYPWWIKDC GITCGGVAST 420

DLPLRTCVYP SYNLGDTDEA VLLASYTWSQ DATRIGSLVK DAPPQPPKED ELVELILQNL 480

ARLHAEHMTY EKIKEAYTGV YHAYCWANDP NVGGAFALFG PGQFSNLYPY LMRPAAGGKF 540

HIVGEASSVH HAWIIGSLES AYTAVYQFLY KYKMWDYLRL LLERWQYGLQ ELETGKHGTA 600

HLQFILGSLP KEYQVKI 617

<210>12

<211>617

<212>PRT

<213> Artificial

<400>12

MDNVDFAESV RTRWARRLIR EKVAKELNIL TERLGEVPGI PPPREGRFLG GGYSHDNLPS 60

DPLYSSIKPA LLKEAPRAEE ELPPRKVCIV GAGVSGLYIA MILDDLKIPN LTYDIFESSS 120

RVGGRLYTHH FTDAKHDYYD IGAMRYPDIP SMKRTFNLFK RTGMPLIKYY LDGENTPQLY 180

NNHFFAKGVV DPYMVSVANG GTVPDDVVDS VGEKLQQAFG YYKEKLAENF DKGFDELMLV 240

DDMTTREYLK RGGPKGEAPK YDFFAIQWME TQNTGTNLFD QAFSESVIDS FDFDNPTKPE 300

WYCIEGGTSL LVDAMKETLV HKVQNNKRVE AISIDLDAPD DGNMSVKIGG KDYSGYSTVF 360

NTTALGCLDR MDLRGLNLHP TQADAIRCLH YDNSTKVALK FSYPWWIKDC GITCGGVAST 420

DLPLRTCVYP SYNLGDTGEA VLLASYTWSQ DATRIGSLVK DAPPQPPKED ELVELILQNL 480

ARLHAEHMTY EKIKEAYTGV YHAYCWANDP NVGGAFALFG PGQFSNLYPY LMRPAAGGKF 540

HIVGEASSVH HAWIIGSLES AYTAVYQFLY KYKMWDYLRL LLERWQYGLQ ELETGKHGTA 600

HLQFILGSLP KEYQVKI 617

<210>13

<211>617

<212>PRT

<213> Artificial

<400>13

MDNVDFAESV RTRWARRLIR EKVAKELNIL TERLGEVPGI PPPREGRFLG GGYSHDNLPS 60

DPLYSSIKPA LLKEAPRAEE ELPPRKVCIV GAGVSGLYIA MILDDLKIPN LTYDIFESSS 120

RVGGRLYTHH FTDAKHDYYD IGAMRYPDIP SMKRTFNLFK RTGMPLIKYY LDGENTPQLY 180

NNHFFAKGVV DPYMVSVANG GTVPDDVVDS VGEKLQQAFG YYKEKLAENF DKGFDELMLV 240

DDMTTREYLK RGGPKGEAPK YDFFAIQWME TQNTGTNLFD QAFSESVIDS FDFDNPTKPE 300

WYCIEGGTSL LVDAMKETLV HKVQNNKRVE AISIDLDAPD DGNMSVKIGG KDYSGYSTVF 360

NTTALGCLDR MDLRGLNLHP TQADAIRCLH YDNSTKVALK FSYPWWIKDC GITCGGAAST 420

DLPLRTCVYP SYNLGDTDEA VLLASYTWSQ DATRIGSLVK DAPPQPPKED ELVELILQNL 480

ARLHAEHMTY EKIKEAYTGV YHAYCWANDP NVGGAFALFG PGQFSNLYPY LMRPAAGGKF 540

HIVGEASSVH HAWIIGSLES AYTAVYQFLY KYKMWDYLRL LLERWQYGLQ ELETGKHGTA 600

HLQFILGSLP KEYQVKI 617

<210>14

<211>617

<212>PRT

<213> Artificial

<400>14

MDNVDFAESV RTRWARRLIR EKVAKELNIL TERLGEVPGI PPPREGRFLG GGYSHDNLPS 60

DPLYSSIKPA LLKEAPRAEE ELPPRKVCIV GAGVSGLYIA MILDDLKIPN LTYDIFESSS 120

RVGGRLYTHH FTDAKHDYYD IGAMRYPDIP SMKRTFNLFK RTGMPLIKYY LDGENTPQLY 180

NNHFFAKGVV DPYMVSVANG GTVPDDVVDS VGEKLQQAFG YYKEKLAEDF DKGFDELMLV 240

DDMTTREYLK RGGPKGEAPK YDFFAIQWME TQNTGTNLFD QAFSESVIDS FDFDNPTKPE 300

WYCIEGGTSL LVDAMKETLV HKVQNNKRVE AISIDLDAPD DGNMSVKIGG KDYSGYSTVF 360

NTTALGCLDR MDLRGLNLHP TQADAIRCLH YDNSTKVALK FSYPWWIKDC GITCGGVAST 420

DLPLRTCVYP SYNLGDTDEA VLLASYTWSQ DATRIGSLVK DAPPQPPKED ELVELILQNL 480

ARLHAEHMTY EKIKEAYTGV YHAYCWANDP NVGGAFALFG PGQFSNLYPY LMRPAAGGKF 540

HIVGEASSVH HAWIIGSLES AYTAVYQFLY KYKMWDYLRL LLERWQYGLQ ELETGKHGTA 600

HLQFILGSLP KEYQVKI 617

<210>15

<211>617

<212>PRT

<213> Artificial

<400>15

MDNVDFAESV RTRWARRLIR EKVAKELNIL TERLGEVPGI PPPREGRFLG GGYSHDNLPS 60

DPLYSSIKPA LLKEAPRAEE ELPPRKVCIV GAGVSGLYIA MILDDLKIPN LTYDIFESSS 120

RTGGRLYTHH FTDAKHDYYD IGAMRYPDIP SMKRTFNLFK RTGMPLIKYY LDGENTPQLY 180

NNHFFAKGVV DPYMVSVANG GTVPDDVVDS VGEKLQQAFG YYKEKLAENF DKGFDELMLV 240

DDMTTREYLK RGGPKGEAPK YDFFAIQWME TQNTGTNLFD QAFSESVIDS FDFDNPTKPE 300

WYCIEGGTSL LVDAMKETLV HKVQNNKRVE AISIDLDAPD DGNMSVKIGG KDYSGYSTVF 360

NTTALGCLDR MDLRGLNLHP TQADAIRCLH YDNSTKVALK FSYPWWIKDC GITCGGVAST 420

DLPLRTCVYP SYNLGDTDEA VLLASYTWSQ DATRIGSLVK DAPPQPPKED ELVELILQNL 480

ARLHAEHMTY EKIKEAYTGV YHAYCWANDP NVGGAFALFG PGQFSNLYPY LMRPAAGGKF 540

HIVGEASSVH HAWIIGSLES AYTAVYQFLY KYKMWDYLRL LLERWQYGLQ ELETGKHGTA 600

HLQFILGSLP KEYQVKI 617

<210>16

<211>617

<212>PRT

<213> Artificial

<400>16

MDNVDFAESV RTRWARRLIR EKVAKELNIL TERLGEVPGI PPPREGRFLG GGYSHDNLPS 60

DPLYSSIKPA LLKEAPRAEE ELPPRKVCIV GAGVSGLYIA MILDDLKIPN LTYDIFESSS 120

RVGGRLYTHH FTDAKHDYYD IGAMRYPDIP SMKRTFNLFK RTGMPLIKYY LDGENTPQLY 180

NNHFFAKGVV DPYMVSVANG GTVPDDVVDS VGEKLQQAFG YYKEKLAENF DKGFDELMLV 240

DDMTTREYLK RGGPKGEAPK YDFFAIQWME TQNTGTNLFD QAFSESVIDS FDFDNPTKPE 300

WYCIEGGTSL LVDAMKETLV HKVQNNKRVE AISIDLDAPD DGNMSVKIGG KDYSGYSTVF 360

NTTALGCLDR MDLRGLNLHP TQADAIRCLH YDNSTKVALK FSYPWWIKDC GITCGGVAST 420

DLPLRTCVYP SYNLGDTDEA VLLASYTWSQ DATRIGSLVK DAPPQPPKED ELVELILQNL 480

ARLHAEHMTY EKIKEAYTGV YHAYCWANDP NVGGAFALFG PGQFSNLYPY LMRPAAGGKF 540

HIVGEASSVH HAWIIGSLES AYTAVYQFLY KYKMWDYLRL LLERWQYGLQ ELETGKHGTA 600

HLQFILGSLP KEYQVKI 617

<210>17

<211>1848

<212>DNA

<213> Artificial

<400>17

atggacaacg tagacttcgc agagtcagta cgaacacgat gggcacgacg actaatccga 60

gagaaggtag caaaggagct aaacattcta acagagcgac taggagaggt accaggaatt 120

ccaccaccac gagagggacg attcctagga ggaggatact cacacgacaa cctaccatca 180

gacccactat actcatcaat taagccagca ctactaaagg aggcaccacg agcagaggag 240

gagctaccac cacgaaaggt atgcattgta ggagcaggag tatcaggact atacattgca 300

atgattctag acgacctaaa gattccaaac ctaacatacg acattttcga gtcatcatca 360

cgaactggag gacgactata cacacaccac ttcacagacg caaagcacga ctactacgac 420

attggagcaa tgcgataccc agacattcca tcaatgaagc gaacattcaa cctattcaag 480

cgaacaggaa tgccactaat taagtactac ctagacggag agaacacacc acagctatac 540

aacaaccact tcttcgcaaa gggagtagta gacccataca tggtatcagt agcaaacgga 600

ggaacagtac cagacgacgt agtagactca gtaggagaga agctacagca ggcattcgga 660

tactacaagg agaagctagc agaggacttc gacaagggat tcgacgagct aatgctagta 720

gacgacatga caacacgaga gtacctaaag cgaggaggac caaagggaga ggcaccaaag 780

tacgacttct tcgcaattca gtggatggag acacagaaca caggaacaaa cctattcgac 840

caggcattct cagagtcagt aattgactca ttcgacttcg acaacccaac aaagccagag 900

tggtactgca ttgagggagg aacatcacta ctagtagacg caatgaagga gacactagta 960

cacaaggtac agaacaacaa gcgagtagag gcaatttcaa ttgacctaga cgcaccagac 1020

gacggaaaca tgtcagtaaa gattggagga aaggactact caggatactc aacagtattc 1080

aacacaacag cactaggatg cctagaccga atggacctac gaggactaaa cctacaccca 1140

acacaggcag acgcaattcg atgcctacac tacgacaact caacaaaggt agcactaaag 1200

ttctcatacc catggtggat taaggactgc ggaattacat gcggaggagc agcatcaaca 1260

gacctaccac tacgaacatg cgtataccca tcatacaacc taggagacac aggcgaggca 1320

gtactactag catcatacac atggtcacag gacgcaacac gaattggatc actagtaaag 1380

gacgcaccac cacagccacc aaaggaggac gagctagtag agctaattct acagaaccta 1440

gcacgactac acgcagagca catgacatac gagaagatta aggaggcata cacaggagta 1500

taccacgcat actgctgggc aaacgaccca aacgtaggag gagcattcgc actattcgga 1560

ccaggacagt tctcaaacct atacccatac ctaatgcgac cagcagcagg aggaaagttc 1620

cacattgtag gagaggcatc atcagtacac cacgcatgga ttattggatc actagagtca 1680

gcatacacag cagtatacca gttcctatac aagtacaaga tgtgggacta cctacgacta 1740

ctactagagc gatggcagta cggactacag gagctagaga caggaaagca cggaacagca 1800

cacctacagt tcattctagg atcactacca aaggagtacc aggtaaag 1848

<210>18

<211>1848

<212>DNA

<213> Artificial

<400>18

atggacaacg tagacttcgc agagtcagta cgaacacgat gggcacgacg actaatccga 60

gagaaggtag caaaggagct aaacattcta acagagcgac taggagaggt accaggaatt 120

ccaccaccac gagagggacg attcctagga ggaggatact cacacgacaa cctaccatca 180

gacccactat actcatcaat taagccagca ctactaaagg aggcaccacg agcagaggag 240

gagctaccac cacgaaaggt atgcattgta ggagcaggag tatcaggact atacattgca 300

atgattctag acgacctaaa gattccaaac ctaacatacg acattttcga gtcatcatca 360

cgaacgggag gacgactata cacacaccac ttcacagacg caaagcacga ctactacgac 420

attggagcaa tgcgataccc agacattcca tcaatgaagc gaacattcaa cctattcaag 480

cgaacaggaa tgccactaat taagtactac ctagacggag agaacacacc acagctatac 540

aacaaccact tcttcgcaaa gggagtagta gacccataca tggtatcagt agcaaacgga 600

ggaacagtac cagacgacgt agtagactca gtaggagaga agctacagca ggcattcgga 660

tactacaagg agaagctagc agagaacttc gacaagggat tcgacgagct aatgctagta 720

gacgacatga caacacgaga gtacctaaag cgaggaggac caaagggaga ggcaccaaag 780

tacgacttct tcgcaattca gtggatggag acacagaaca caggaacaaa cctattcgac 840

caggcattct cagagtcagt aattgactca ttcgacttcg acaacccaac aaagccagag 900

tggtactgca ttgagggagg aacatcacta ctagtagacg caatgaagga gacactagta 960

cacaaggtac agaacaacaa gcgagtagag gcaatttcaa ttgacctaga cgcaccagac 1020

gacggaaaca tgtcagtaaa gattggagga aaggactact caggatactc aacagtattc 1080

aacacaacag cactaggatg cctagaccga atggacctac gaggactaaa cctacaccca 1140

acacaggcag acgcaattcg atgcctacac tacgacaact caacaaaggt agcactaaag 1200

ttctcatacc catggtggat taaggactgc ggaattacat gcggaggagc agcatcaaca 1260

gacctaccac tacgaacatg cgtataccca tcatacaacc taggagacac aggcgaggca 1320

gtactactag catcatacac atggtcacag gacgcaacac gaattggatc actagtaaag 1380

gacgcaccac cacagccacc aaaggaggac gagctagtag agctaattct acagaaccta 1440

gcacgactac acgcagagca catgacatac gagaagatta aggaggcata cacaggagta 1500

taccacgcat actgctgggc aaacgaccca aacgtaggag gagcattcgc actattcgga 1560

ccaggacagt tctcaaacct atacccatac ctaatgcgac cagcagcagg aggaaagttc 1620

cacattgtag gagaggcatc atcagtacac cacgcatgga ttattggatc actagagtca 1680

gcatacacag cagtatacca gttcctatac aagtacaaga tgtgggacta cctacgacta 1740

ctactagagc gatggcagta cggactacag gagctagaga caggaaagca cggaacagca 1800

cacctacagt tcattctagg atcactacca aaggagtacc aggtaaag 1848

<210>19

<211>1848

<212>DNA

<213> Artificial

<400>19

atggacaacg tagacttcgc agagtcagta cgaacacgat gggcacgacg actaatccga 60

gagaaggtag caaaggagct aaacattcta acagagcgac taggagaggt accaggaatt 120

ccaccaccac gagagggacg attcctagga ggaggatact cacacgacaa cctaccatca 180

gacccactat actcatcaat taagccagca ctactaaagg aggcaccacg agcagaggag 240

gagctaccac cacgaaaggt atgcattgta ggagcaggag tatcaggact atacattgca 300

atgattctag acgacctaaa gattccaaac ctaacatacg acattttcga gtcatcatca 360

cgaacgggag gacgactata cacacaccac ttcacagacg caaagcacga ctactacgac 420

attggagcaa tgcgataccc agacattcca tcaatgaagc gaacattcaa cctattcaag 480

cgaacaggaa tgccactaat taagtactac ctagacggag agaacacacc acagctatac 540

aacaaccact tcttcgcaaa gggagtagta gacccataca tggtatcagt agcaaacgga 600

ggaacagtac cagacgacgt agtagactca gtaggagaga agctacagca ggcattcgga 660

tactacaagg agaagctagc agaggacttc gacaagggat tcgacgagct aatgctagta 720

gacgacatga caacacgaga gtacctaaag cgaggaggac caaagggaga ggcaccaaag 780

tacgacttct tcgcaattca gtggatggag acacagaaca caggaacaaa cctattcgac 840

caggcattct cagagtcagt aattgactca ttcgacttcg acaacccaac aaagccagag 900

tggtactgca ttgagggagg aacatcacta ctagtagacg caatgaagga gacactagta 960

cacaaggtac agaacaacaa gcgagtagag gcaatttcaa ttgacctaga cgcaccagac 1020

gacggaaaca tgtcagtaaa gattggagga aaggactact caggatactc aacagtattc 1080

aacacaacag cactaggatg cctagaccga atggacctac gaggactaaa cctacaccca 1140

acacaggcag acgcaattcg atgcctacac tacgacaact caacaaaggt agcactaaag 1200

ttctcatacc catggtggat taaggactgc ggaattacat gcggaggagt agcatcaaca 1260

gacctaccac tacgaacatg cgtataccca tcatacaacc taggagacac aggcgaggca 1320

gtactactag catcatacac atggtcacag gacgcaacac gaattggatc actagtaaag 1380

gacgcaccac cacagccacc aaaggaggac gagctagtag agctaattct acagaaccta 1440

gcacgactac acgcagagca catgacatac gagaagatta aggaggcata cacaggagta 1500

taccacgcat actgctgggc aaacgaccca aacgtaggag gagcattcgc actattcgga 1560

ccaggacagt tctcaaacct atacccatac ctaatgcgac cagcagcagg aggaaagttc 1620

cacattgtag gagaggcatc atcagtacac cacgcatgga ttattggatc actagagtca 1680

gcatacacag cagtatacca gttcctatac aagtacaaga tgtgggacta cctacgacta 1740

ctactagagc gatggcagta cggactacag gagctagaga caggaaagca cggaacagca 1800

cacctacagt tcattctagg atcactacca aaggagtacc aggtaaag 1848

<210>20

<211>1848

<212>DNA

<213> Artificial

<400>20

atggacaacg tagacttcgc agagtcagta cgaacacgat gggcacgacg actaatccga 60

gagaaggtag caaaggagct aaacattcta acagagcgac taggagaggt accaggaatt 120

ccaccaccac gagagggacg attcctagga ggaggatact cacacgacaa cctaccatca 180

gacccactat actcatcaat taagccagca ctactaaagg aggcaccacg agcagaggag 240

gagctaccac cacgaaaggt atgcattgta ggagcaggag tatcaggact atacattgca 300

atgattctag acgacctaaa gattccaaac ctaacatacg acattttcga gtcatcatca 360

cgaacgggag gacgactata cacacaccac ttcacagacg caaagcacga ctactacgac 420

attggagcaa tgcgataccc agacattcca tcaatgaagc gaacattcaa cctattcaag 480

cgaacaggaa tgccactaat taagtactac ctagacggag agaacacacc acagctatac 540

aacaaccact tcttcgcaaa gggagtagta gacccataca tggtatcagt agcaaacgga 600

ggaacagtac cagacgacgt agtagactca gtaggagaga agctacagca ggcattcgga 660

tactacaagg agaagctagc agaggacttc gacaagggat tcgacgagct aatgctagta 720

gacgacatga caacacgaga gtacctaaag cgaggaggac caaagggaga ggcaccaaag 780

tacgacttct tcgcaattca gtggatggag acacagaaca caggaacaaa cctattcgac 840

caggcattct cagagtcagt aattgactca ttcgacttcg acaacccaac aaagccagag 900

tggtactgca ttgagggagg aacatcacta ctagtagacg caatgaagga gacactagta 960

cacaaggtac agaacaacaa gcgagtagag gcaatttcaa ttgacctaga cgcaccagac 1020

gacggaaaca tgtcagtaaa gattggagga aaggactact caggatactc aacagtattc 1080

aacacaacag cactaggatg cctagaccga atggacctac gaggactaaa cctacaccca 1140

acacaggcag acgcaattcg atgcctacac tacgacaact caacaaaggt agcactaaag 1200

ttctcatacc catggtggat taaggactgc ggaattacat gcggaggagc agcatcaaca 1260

gacctaccac tacgaacatg cgtataccca tcatacaacc taggagacac agacgaggca 1320

gtactactag catcatacac atggtcacag gacgcaacac gaattggatc actagtaaag 1380

gacgcaccac cacagccacc aaaggaggac gagctagtag agctaattct acagaaccta 1440

gcacgactac acgcagagca catgacatac gagaagatta aggaggcata cacaggagta 1500

taccacgcat actgctgggc aaacgaccca aacgtaggag gagcattcgc actattcgga 1560

ccaggacagt tctcaaacct atacccatac ctaatgcgac cagcagcagg aggaaagttc 1620

cacattgtag gagaggcatc atcagtacac cacgcatgga ttattggatc actagagtca 1680

gcatacacag cagtatacca gttcctatac aagtacaaga tgtgggacta cctacgacta 1740

ctactagagc gatggcagta cggactacag gagctagaga caggaaagca cggaacagca 1800

cacctacagt tcattctagg atcactacca aaggagtacc aggtaaag 1848

<210>21

<211>1848

<212>DNA

<213> Artificial

<400>21

atggacaacg tagacttcgc agagtcagta cgaacacgat gggcacgacg actaatccga 60

gagaaggtag caaaggagct aaacattcta acagagcgac taggagaggt accaggaatt 120

ccaccaccac gagagggacg attcctagga ggaggatact cacacgacaa cctaccatca 180

gacccactat actcatcaat taagccagca ctactaaagg aggcaccacg agcagaggag 240

gagctaccac cacgaaaggt atgcattgta ggagcaggag tatcaggact atacattgca 300

atgattctag acgacctaaa gattccaaac ctaacatacg acattttcga gtcatcatca 360

cgaactggag gacgactata cacacaccac ttcacagacg caaagcacga ctactacgac 420

attggagcaa tgcgataccc agacattcca tcaatgaagc gaacattcaa cctattcaag 480

cgaacaggaa tgccactaat taagtactac ctagacggag agaacacacc acagctatac 540

aacaaccact tcttcgcaaa gggagtagta gacccataca tggtatcagt agcaaacgga 600

ggaacagtac cagacgacgt agtagactca gtaggagaga agctacagca ggcattcgga 660

tactacaagg agaagctagc agagaacttc gacaagggat tcgacgagct aatgctagta 720

gacgacatga caacacgaga gtacctaaag cgaggaggac caaagggaga ggcaccaaag 780

tacgacttct tcgcaattca gtggatggag acacagaaca caggaacaaa cctattcgac 840

caggcattct cagagtcagt aattgactca ttcgacttcg acaacccaac aaagccagag 900

tggtactgca ttgagggagg aacatcacta ctagtagacg caatgaagga gacactagta 960

cacaaggtac agaacaacaa gcgagtagag gcaatttcaa ttgacctaga cgcaccagac 1020

gacggaaaca tgtcagtaaa gattggagga aaggactact caggatactc aacagtattc 1080

aacacaacag cactaggatg cctagaccga atggacctac gaggactaaa cctacaccca 1140

acacaggcag acgcaattcg atgcctacac tacgacaact caacaaaggt agcactaaag 1200

ttctcatacc catggtggat taaggactgc ggaattacat gcggaggagc agcatcaaca 1260

gacctaccac tacgaacatg cgtataccca tcatacaacc taggagacac aggcgaggca 1320

gtactactag catcatacac atggtcacag gacgcaacac gaattggatc actagtaaag 1380

gacgcaccac cacagccacc aaaggaggac gagctagtag agctaattct acagaaccta 1440

gcacgactac acgcagagca catgacatac gagaagatta aggaggcata cacaggagta 1500

taccacgcat actgctgggc aaacgaccca aacgtaggag gagcattcgc actattcgga 1560

ccaggacagt tctcaaacct atacccatac ctaatgcgac cagcagcagg aggaaagttc 1620

cacattgtag gagaggcatc atcagtacac cacgcatgga ttattggatc actagagtca 1680

gcatacacag cagtatacca gttcctatac aagtacaaga tgtgggacta cctacgacta 1740

ctactagagc gatggcagta cggactacag gagctagaga caggaaagca cggaacagca 1800

cacctacagt tcattctagg atcactacca aaggagtacc aggtaaag 1848

<210>22

<211>1848

<212>DNA

<213> Artificial

<400>22

atggacaacg tagacttcgc agagtcagta cgaacacgat gggcacgacg actaatccga 60

gagaaggtag caaaggagct aaacattcta acagagcgac taggagaggt accaggaatt 120

ccaccaccac gagagggacg attcctagga ggaggatact cacacgacaa cctaccatca 180

gacccactat actcatcaat taagccagca ctactaaagg aggcaccacg agcagaggag 240

gagctaccac cacgaaaggt atgcattgta ggagcaggag tatcaggact atacattgca 300

atgattctag acgacctaaa gattccaaac ctaacatacg acattttcga gtcatcatca 360

cgaactggag gacgactata cacacaccac ttcacagacg caaagcacga ctactacgac 420

attggagcaa tgcgataccc agacattcca tcaatgaagc gaacattcaa cctattcaag 480

cgaacaggaa tgccactaat taagtactac ctagacggag agaacacacc acagctatac 540

aacaaccact tcttcgcaaa gggagtagta gacccataca tggtatcagt agcaaacgga 600

ggaacagtac cagacgacgt agtagactca gtaggagaga agctacagca ggcattcgga 660

tactacaagg agaagctagc agaggacttc gacaagggat tcgacgagct aatgctagta 720

gacgacatga caacacgaga gtacctaaag cgaggaggac caaagggaga ggcaccaaag 780

tacgacttct tcgcaattca gtggatggag acacagaaca caggaacaaa cctattcgac 840

caggcattct cagagtcagt aattgactca ttcgacttcg acaacccaac aaagccagag 900

tggtactgca ttgagggagg aacatcacta ctagtagacg caatgaagga gacactagta 960

cacaaggtac agaacaacaa gcgagtagag gcaatttcaa ttgacctaga cgcaccagac 1020

gacggaaaca tgtcagtaaa gattggagga aaggactact caggatactc aacagtattc 1080

aacacaacag cactaggatg cctagaccga atggacctac gaggactaaa cctacaccca 1140

acacaggcag acgcaattcg atgcctacac tacgacaact caacaaaggt agcactaaag 1200

ttctcatacc catggtggat taaggactgc ggaattacat gcggaggagt agcatcaaca 1260

gacctaccac tacgaacatg cgtataccca tcatacaacc taggagacac aggcgaggca 1320

gtactactag catcatacac atggtcacag gacgcaacac gaattggatc actagtaaag 1380

gacgcaccac cacagccacc aaaggaggac gagctagtag agctaattct acagaaccta 1440

gcacgactac acgcagagca catgacatac gagaagatta aggaggcata cacaggagta 1500

taccacgcat actgctgggc aaacgaccca aacgtaggag gagcattcgc actattcgga 1560

ccaggacagt tctcaaacct atacccatac ctaatgcgac cagcagcagg aggaaagttc 1620

cacattgtag gagaggcatc atcagtacac cacgcatgga ttattggatc actagagtca 1680

gcatacacag cagtatacca gttcctatac aagtacaaga tgtgggacta cctacgacta 1740

ctactagagc gatggcagta cggactacag gagctagaga caggaaagca cggaacagca 1800

cacctacagt tcattctagg atcactacca aaggagtacc aggtaaag 1848

<210>23

<211>1848

<212>DNA

<213> Artificial

<400>23

atggacaacg tagacttcgc agagtcagta cgaacacgat gggcacgacg actaatccga 60

gagaaggtag caaaggagct aaacattcta acagagcgac taggagaggt accaggaatt 120

ccaccaccac gagagggacg attcctagga ggaggatact cacacgacaa cctaccatca 180

gacccactat actcatcaat taagccagca ctactaaagg aggcaccacg agcagaggag 240

gagctaccac cacgaaaggt atgcattgta ggagcaggag tatcaggact atacattgca 300

atgattctag acgacctaaa gattccaaac ctaacatacg acattttcga gtcatcatca 360

cgaactggag gacgactata cacacaccac ttcacagacg caaagcacga ctactacgac 420

attggagcaa tgcgataccc agacattcca tcaatgaagc gaacattcaa cctattcaag 480

cgaacaggaa tgccactaat taagtactac ctagacggag agaacacacc acagctatac 540

aacaaccact tcttcgcaaa gggagtagta gacccataca tggtatcagt agcaaacgga 600

ggaacagtac cagacgacgt agtagactca gtaggagaga agctacagca ggcattcgga 660

tactacaagg agaagctagc agaggacttc gacaagggat tcgacgagct aatgctagta 720

gacgacatga caacacgaga gtacctaaag cgaggaggac caaagggaga ggcaccaaag 780

tacgacttct tcgcaattca gtggatggag acacagaaca caggaacaaa cctattcgac 840

caggcattct cagagtcagt aattgactca ttcgacttcg acaacccaac aaagccagag 900

tggtactgca ttgagggagg aacatcacta ctagtagacg caatgaagga gacactagta 960

cacaaggtac agaacaacaa gcgagtagag gcaatttcaa ttgacctaga cgcaccagac 1020

gacggaaaca tgtcagtaaa gattggagga aaggactact caggatactc aacagtattc 1080

aacacaacag cactaggatg cctagaccga atggacctac gaggactaaa cctacaccca 1140

acacaggcag acgcaattcg atgcctacac tacgacaact caacaaaggt agcactaaag 1200

ttctcatacc catggtggat taaggactgc ggaattacat gcggaggagc agcatcaaca 1260

gacctaccac tacgaacatg cgtataccca tcatacaacc taggagacac agacgaggca 1320

gtactactag catcatacac atggtcacag gacgcaacac gaattggatc actagtaaag 1380

gacgcaccac cacagccacc aaaggaggac gagctagtag agctaattct acagaaccta 1440

gcacgactac acgcagagca catgacatac gagaagatta aggaggcata cacaggagta 1500

taccacgcat actgctgggc aaacgaccca aacgtaggag gagcattcgc actattcgga 1560

ccaggacagt tctcaaacct atacccatac ctaatgcgac cagcagcagg aggaaagttc 1620

cacattgtag gagaggcatc atcagtacac cacgcatgga ttattggatc actagagtca 1680

gcatacacag cagtatacca gttcctatac aagtacaaga tgtgggacta cctacgacta 1740

ctactagagc gatggcagta cggactacag gagctagaga caggaaagca cggaacagca 1800

cacctacagt tcattctagg atcactacca aaggagtacc aggtaaag 1848

<210>24

<211>1848

<212>DNA

<213> Artificial

<400>24

atggacaacg tagacttcgc agagtcagta cgaacacgat gggcacgacg actaatccga 60

gagaaggtag caaaggagct aaacattcta acagagcgac taggagaggt accaggaatt 120

ccaccaccac gagagggacg attcctagga ggaggatact cacacgacaa cctaccatca 180

gacccactat actcatcaat taagccagca ctactaaagg aggcaccacg agcagaggag 240

gagctaccac cacgaaaggt atgcattgta ggagcaggag tatcaggact atacattgca 300

atgattctag acgacctaaa gattccaaac ctaacatacg acattttcga gtcatcatca 360

cgaacgggag gacgactata cacacaccac ttcacagacg caaagcacga ctactacgac 420

attggagcaa tgcgataccc agacattcca tcaatgaagc gaacattcaa cctattcaag 480

cgaacaggaa tgccactaat taagtactac ctagacggag agaacacacc acagctatac 540

aacaaccact tcttcgcaaa gggagtagta gacccataca tggtatcagt agcaaacgga 600

ggaacagtac cagacgacgt agtagactca gtaggagaga agctacagca ggcattcgga 660

tactacaagg agaagctagc agagaacttc gacaagggat tcgacgagct aatgctagta 720

gacgacatga caacacgaga gtacctaaag cgaggaggac caaagggaga ggcaccaaag 780

tacgacttct tcgcaattca gtggatggag acacagaaca caggaacaaa cctattcgac 840

caggcattct cagagtcagt aattgactca ttcgacttcg acaacccaac aaagccagag 900

tggtactgca ttgagggagg aacatcacta ctagtagacg caatgaagga gacactagta 960

cacaaggtac agaacaacaa gcgagtagag gcaatttcaa ttgacctaga cgcaccagac 1020

gacggaaaca tgtcagtaaa gattggagga aaggactact caggatactc aacagtattc 1080

aacacaacag cactaggatg cctagaccga atggacctac gaggactaaa cctacaccca 1140

acacaggcag acgcaattcg atgcctacac tacgacaact caacaaaggt agcactaaag 1200

ttctcatacc catggtggat taaggactgc ggaattacat gcggaggagt agcatcaaca 1260

gacctaccac tacgaacatg cgtataccca tcatacaacc taggagacac aggcgaggca 1320

gtactactag catcatacac atggtcacag gacgcaacac gaattggatc actagtaaag 1380

gacgcaccac cacagccacc aaaggaggac gagctagtag agctaattct acagaaccta 1440

gcacgactac acgcagagca catgacatac gagaagatta aggaggcata cacaggagta 1500

taccacgcat actgctgggc aaacgaccca aacgtaggag gagcattcgc actattcgga 1560

ccaggacagt tctcaaacct atacccatac ctaatgcgac cagcagcagg aggaaagttc 1620

cacattgtag gagaggcatc atcagtacac cacgcatgga ttattggatc actagagtca 1680

gcatacacag cagtatacca gttcctatac aagtacaaga tgtgggacta cctacgacta 1740

ctactagagc gatggcagta cggactacag gagctagaga caggaaagca cggaacagca 1800

cacctacagt tcattctagg atcactacca aaggagtacc aggtaaag 1848

<210>25

<211>1848

<212>DNA

<213> Artificial

<400>25

atggacaacg tagacttcgc agagtcagta cgaacacgat gggcacgacg actaatccga 60

gagaaggtag caaaggagct aaacattcta acagagcgac taggagaggt accaggaatt 120

ccaccaccac gagagggacg attcctagga ggaggatact cacacgacaa cctaccatca 180

gacccactat actcatcaat taagccagca ctactaaagg aggcaccacg agcagaggag 240

gagctaccac cacgaaaggt atgcattgta ggagcaggag tatcaggact atacattgca 300

atgattctag acgacctaaa gattccaaac ctaacatacg acattttcga gtcatcatca 360

cgaacgggag gacgactata cacacaccac ttcacagacg caaagcacga ctactacgac 420

attggagcaa tgcgataccc agacattcca tcaatgaagc gaacattcaa cctattcaag 480

cgaacaggaa tgccactaat taagtactac ctagacggag agaacacacc acagctatac 540

aacaaccact tcttcgcaaa gggagtagta gacccataca tggtatcagt agcaaacgga 600

ggaacagtac cagacgacgt agtagactca gtaggagaga agctacagca ggcattcgga 660

tactacaagg agaagctagc agagaacttc gacaagggat tcgacgagct aatgctagta 720

gacgacatga caacacgaga gtacctaaag cgaggaggac caaagggaga ggcaccaaag 780

tacgacttct tcgcaattca gtggatggag acacagaaca caggaacaaa cctattcgac 840

caggcattct cagagtcagt aattgactca ttcgacttcg acaacccaac aaagccagag 900

tggtactgca ttgagggagg aacatcacta ctagtagacg caatgaagga gacactagta 960

cacaaggtac agaacaacaa gcgagtagag gcaatttcaa ttgacctaga cgcaccagac 1020

gacggaaaca tgtcagtaaa gattggagga aaggactact caggatactc aacagtattc 1080

aacacaacag cactaggatg cctagaccga atggacctac gaggactaaa cctacaccca 1140

acacaggcag acgcaattcg atgcctacac tacgacaact caacaaaggt agcactaaag 1200

ttctcatacc catggtggat taaggactgc ggaattacat gcggaggagc agcatcaaca 1260

gacctaccac tacgaacatg cgtataccca tcatacaacc taggagacac agacgaggca 1320

gtactactag catcatacac atggtcacag gacgcaacac gaattggatc actagtaaag 1380

gacgcaccac cacagccacc aaaggaggac gagctagtag agctaattct acagaaccta 1440

gcacgactac acgcagagca catgacatac gagaagatta aggaggcata cacaggagta 1500

taccacgcat actgctgggc aaacgaccca aacgtaggag gagcattcgc actattcgga 1560

ccaggacagt tctcaaacct atacccatac ctaatgcgac cagcagcagg aggaaagttc 1620

cacattgtag gagaggcatc atcagtacac cacgcatgga ttattggatc actagagtca 1680

gcatacacag cagtatacca gttcctatac aagtacaaga tgtgggacta cctacgacta 1740

ctactagagc gatggcagta cggactacag gagctagaga caggaaagca cggaacagca 1800

cacctacagt tcattctagg atcactacca aaggagtacc aggtaaag 1848

<210>26

<211>1848

<212>DNA

<213> Artificial

<400>26

atggacaacg tagacttcgc agagtcagta cgaacacgat gggcacgacg actaatccga 60

gagaaggtag caaaggagct aaacattcta acagagcgac taggagaggt accaggaatt 120

ccaccaccac gagagggacg attcctagga ggaggatact cacacgacaa cctaccatca 180

gacccactat actcatcaat taagccagca ctactaaagg aggcaccacg agcagaggag 240

gagctaccac cacgaaaggt atgcattgta ggagcaggag tatcaggact atacattgca 300

atgattctag acgacctaaa gattccaaac ctaacatacg acattttcga gtcatcatca 360

cgaacgggag gacgactata cacacaccac ttcacagacg caaagcacga ctactacgac 420

attggagcaa tgcgataccc agacattcca tcaatgaagc gaacattcaa cctattcaag 480

cgaacaggaa tgccactaat taagtactac ctagacggag agaacacacc acagctatac 540

aacaaccact tcttcgcaaa gggagtagta gacccataca tggtatcagt agcaaacgga 600

ggaacagtac cagacgacgt agtagactca gtaggagaga agctacagca ggcattcgga 660

tactacaagg agaagctagc agaggacttc gacaagggat tcgacgagct aatgctagta 720

gacgacatga caacacgaga gtacctaaag cgaggaggac caaagggaga ggcaccaaag 780

tacgacttct tcgcaattca gtggatggag acacagaaca caggaacaaa cctattcgac 840

caggcattct cagagtcagt aattgactca ttcgacttcg acaacccaac aaagccagag 900

tggtactgca ttgagggagg aacatcacta ctagtagacg caatgaagga gacactagta 960

cacaaggtac agaacaacaa gcgagtagag gcaatttcaa ttgacctaga cgcaccagac 1020

gacggaaaca tgtcagtaaa gattggagga aaggactact caggatactc aacagtattc 1080

aacacaacag cactaggatg cctagaccga atggacctac gaggactaaa cctacaccca 1140

acacaggcag acgcaattcg atgcctacac tacgacaact caacaaaggt agcactaaag 1200

ttctcatacc catggtggat taaggactgc ggaattacat gcggaggagt agcatcaaca 1260

gacctaccac tacgaacatg cgtataccca tcatacaacc taggagacac agacgaggca 1320

gtactactag catcatacac atggtcacag gacgcaacac gaattggatc actagtaaag 1380

gacgcaccac cacagccacc aaaggaggac gagctagtag agctaattct acagaaccta 1440

gcacgactac acgcagagca catgacatac gagaagatta aggaggcata cacaggagta 1500

taccacgcat actgctgggc aaacgaccca aacgtaggag gagcattcgc actattcgga 1560

ccaggacagt tctcaaacct atacccatac ctaatgcgac cagcagcagg aggaaagttc 1620

cacattgtag gagaggcatc atcagtacac cacgcatgga ttattggatc actagagtca 1680

gcatacacag cagtatacca gttcctatac aagtacaaga tgtgggacta cctacgacta 1740

ctactagagc gatggcagta cggactacag gagctagaga caggaaagca cggaacagca 1800

cacctacagt tcattctagg atcactacca aaggagtacc aggtaaag 1848

<210>27

<211>1848

<212>DNA

<213> Artificial

<400>27

atggacaacg tagacttcgc agagtcagta cgaacacgat gggcacgacg actaatccga 60

gagaaggtag caaaggagct aaacattcta acagagcgac taggagaggt accaggaatt 120

ccaccaccac gagagggacg attcctagga ggaggatact cacacgacaa cctaccatca 180

gacccactat actcatcaat taagccagca ctactaaagg aggcaccacg agcagaggag 240

gagctaccac cacgaaaggt atgcattgta ggagcaggag tatcaggact atacattgca 300

atgattctag acgacctaaa gattccaaac ctaacatacg acattttcga gtcatcatca 360

cgaactggag gacgactata cacacaccac ttcacagacg caaagcacga ctactacgac 420

attggagcaa tgcgataccc agacattcca tcaatgaagc gaacattcaa cctattcaag 480

cgaacaggaa tgccactaat taagtactac ctagacggag agaacacacc acagctatac 540

aacaaccact tcttcgcaaa gggagtagta gacccataca tggtatcagt agcaaacgga 600

ggaacagtac cagacgacgt agtagactca gtaggagaga agctacagca ggcattcgga 660

tactacaagg agaagctagc agagaacttc gacaagggat tcgacgagct aatgctagta 720

gacgacatga caacacgaga gtacctaaag cgaggaggac caaagggaga ggcaccaaag 780

tacgacttct tcgcaattca gtggatggag acacagaaca caggaacaaa cctattcgac 840

caggcattct cagagtcagt aattgactca ttcgacttcg acaacccaac aaagccagag 900

tggtactgca ttgagggagg aacatcacta ctagtagacg caatgaagga gacactagta 960

cacaaggtac agaacaacaa gcgagtagag gcaatttcaa ttgacctaga cgcaccagac 1020

gacggaaaca tgtcagtaaa gattggagga aaggactact caggatactc aacagtattc 1080

aacacaacag cactaggatg cctagaccga atggacctac gaggactaaa cctacaccca 1140

acacaggcag acgcaattcg atgcctacac tacgacaact caacaaaggt agcactaaag 1200

ttctcatacc catggtggat taaggactgc ggaattacat gcggaggagt agcatcaaca 1260

gacctaccac tacgaacatg cgtataccca tcatacaacc taggagacac aggcgaggca 1320

gtactactag catcatacac atggtcacag gacgcaacac gaattggatc actagtaaag 1380

gacgcaccac cacagccacc aaaggaggac gagctagtag agctaattct acagaaccta 1440

gcacgactac acgcagagca catgacatac gagaagatta aggaggcata cacaggagta 1500

taccacgcat actgctgggc aaacgaccca aacgtaggag gagcattcgc actattcgga 1560

ccaggacagt tctcaaacct atacccatac ctaatgcgac cagcagcagg aggaaagttc 1620

cacattgtag gagaggcatc atcagtacac cacgcatgga ttattggatc actagagtca 1680

gcatacacag cagtatacca gttcctatac aagtacaaga tgtgggacta cctacgacta 1740

ctactagagc gatggcagta cggactacag gagctagaga caggaaagca cggaacagca 1800

cacctacagt tcattctagg atcactacca aaggagtacc aggtaaag 1848

<210>28

<211>1848

<212>DNA

<213> Artificial

<400>28

atggacaacg tagacttcgc agagtcagta cgaacacgat gggcacgacg actaatccga 60

gagaaggtag caaaggagct aaacattcta acagagcgac taggagaggt accaggaatt 120

ccaccaccac gagagggacg attcctagga ggaggatact cacacgacaa cctaccatca 180

gacccactat actcatcaat taagccagca ctactaaagg aggcaccacg agcagaggag 240

gagctaccac cacgaaaggt atgcattgta ggagcaggag tatcaggact atacattgca 300

atgattctag acgacctaaa gattccaaac ctaacatacg acattttcga gtcatcatca 360

cgaactggag gacgactata cacacaccac ttcacagacg caaagcacga ctactacgac 420

attggagcaa tgcgataccc agacattcca tcaatgaagc gaacattcaa cctattcaag 480

cgaacaggaa tgccactaat taagtactac ctagacggag agaacacacc acagctatac 540

aacaaccact tcttcgcaaa gggagtagta gacccataca tggtatcagt agcaaacgga 600

ggaacagtac cagacgacgt agtagactca gtaggagaga agctacagca ggcattcgga 660

tactacaagg agaagctagc agagaacttc gacaagggat tcgacgagct aatgctagta 720

gacgacatga caacacgaga gtacctaaag cgaggaggac caaagggaga ggcaccaaag 780

tacgacttct tcgcaattca gtggatggag acacagaaca caggaacaaa cctattcgac 840

caggcattct cagagtcagt aattgactca ttcgacttcg acaacccaac aaagccagag 900

tggtactgca ttgagggagg aacatcacta ctagtagacg caatgaagga gacactagta 960

cacaaggtac agaacaacaa gcgagtagag gcaatttcaa ttgacctaga cgcaccagac 1020

gacggaaaca tgtcagtaaa gattggagga aaggactact caggatactc aacagtattc 1080

aacacaacag cactaggatg cctagaccga atggacctac gaggactaaa cctacaccca 1140

acacaggcag acgcaattcg atgcctacac tacgacaact caacaaaggt agcactaaag 1200

ttctcatacc catggtggat taaggactgc ggaattacat gcggaggagc agcatcaaca 1260

gacctaccac tacgaacatg cgtataccca tcatacaacc taggagacac agacgaggca 1320

gtactactag catcatacac atggtcacag gacgcaacac gaattggatc actagtaaag 1380

gacgcaccac cacagccacc aaaggaggac gagctagtag agctaattct acagaaccta 1440

gcacgactac acgcagagca catgacatac gagaagatta aggaggcata cacaggagta 1500

taccacgcat actgctgggc aaacgaccca aacgtaggag gagcattcgc actattcgga 1560

ccaggacagt tctcaaacct atacccatac ctaatgcgac cagcagcagg aggaaagttc 1620

cacattgtag gagaggcatc atcagtacac cacgcatgga ttattggatc actagagtca 1680

gcatacacag cagtatacca gttcctatac aagtacaaga tgtgggacta cctacgacta 1740

ctactagagc gatggcagta cggactacag gagctagaga caggaaagca cggaacagca 1800

cacctacagt tcattctagg atcactacca aaggagtacc aggtaaag 1848

<210>29

<211>1848

<212>DNA

<213> Artificial

<400>29

atggacaacg tagacttcgc agagtcagta cgaacacgat gggcacgacg actaatccga 60

gagaaggtag caaaggagct aaacattcta acagagcgac taggagaggt accaggaatt 120

ccaccaccac gagagggacg attcctagga ggaggatact cacacgacaa cctaccatca 180

gacccactat actcatcaat taagccagca ctactaaagg aggcaccacg agcagaggag 240

gagctaccac cacgaaaggt atgcattgta ggagcaggag tatcaggact atacattgca 300

atgattctag acgacctaaa gattccaaac ctaacatacg acattttcga gtcatcatca 360

cgaactggag gacgactata cacacaccac ttcacagacg caaagcacga ctactacgac 420

attggagcaa tgcgataccc agacattcca tcaatgaagc gaacattcaa cctattcaag 480

cgaacaggaa tgccactaat taagtactac ctagacggag agaacacacc acagctatac 540

aacaaccact tcttcgcaaa gggagtagta gacccataca tggtatcagt agcaaacgga 600

ggaacagtac cagacgacgt agtagactca gtaggagaga agctacagca ggcattcgga 660

tactacaagg agaagctagc agaggacttc gacaagggat tcgacgagct aatgctagta 720

gacgacatga caacacgaga gtacctaaag cgaggaggac caaagggaga ggcaccaaag 780

tacgacttct tcgcaattca gtggatggag acacagaaca caggaacaaa cctattcgac 840

caggcattct cagagtcagt aattgactca ttcgacttcg acaacccaac aaagccagag 900

tggtactgca ttgagggagg aacatcacta ctagtagacg caatgaagga gacactagta 960

cacaaggtac agaacaacaa gcgagtagag gcaatttcaa ttgacctaga cgcaccagac 1020

gacggaaaca tgtcagtaaa gattggagga aaggactact caggatactc aacagtattc 1080

aacacaacag cactaggatg cctagaccga atggacctac gaggactaaa cctacaccca 1140

acacaggcag acgcaattcg atgcctacac tacgacaact caacaaaggt agcactaaag 1200

ttctcatacc catggtggat taaggactgc ggaattacat gcggaggagt agcatcaaca 1260

gacctaccac tacgaacatg cgtataccca tcatacaacc taggagacac agacgaggca 1320

gtactactag catcatacac atggtcacag gacgcaacac gaattggatc actagtaaag 1380

gacgcaccac cacagccacc aaaggaggac gagctagtag agctaattct acagaaccta 1440

gcacgactac acgcagagca catgacatac gagaagatta aggaggcata cacaggagta 1500

taccacgcat actgctgggc aaacgaccca aacgtaggag gagcattcgc actattcgga 1560

ccaggacagt tctcaaacct atacccatac ctaatgcgac cagcagcagg aggaaagttc 1620

cacattgtag gagaggcatc atcagtacac cacgcatgga ttattggatc actagagtca 1680

gcatacacag cagtatacca gttcctatac aagtacaaga tgtgggacta cctacgacta 1740

ctactagagc gatggcagta cggactacag gagctagaga caggaaagca cggaacagca 1800

cacctacagt tcattctagg atcactacca aaggagtacc aggtaaag 1848

<210>30

<211>1848

<212>DNA

<213> Artificial

<400>30

atggacaacg tagacttcgc agagtcagta cgaacacgat gggcacgacg actaatccga 60

gagaaggtag caaaggagct aaacattcta acagagcgac taggagaggt accaggaatt 120

ccaccaccac gagagggacg attcctagga ggaggatact cacacgacaa cctaccatca 180

gacccactat actcatcaat taagccagca ctactaaagg aggcaccacg agcagaggag 240

gagctaccac cacgaaaggt atgcattgta ggagcaggag tatcaggact atacattgca 300

atgattctag acgacctaaa gattccaaac ctaacatacg acattttcga gtcatcatca 360

cgaacgggag gacgactata cacacaccac ttcacagacg caaagcacga ctactacgac 420

attggagcaa tgcgataccc agacattcca tcaatgaagc gaacattcaa cctattcaag 480

cgaacaggaa tgccactaat taagtactac ctagacggag agaacacacc acagctatac 540

aacaaccact tcttcgcaaa gggagtagta gacccataca tggtatcagt agcaaacgga 600

ggaacagtac cagacgacgt agtagactca gtaggagaga agctacagca ggcattcgga 660

tactacaagg agaagctagc agagaacttc gacaagggat tcgacgagct aatgctagta 720

gacgacatga caacacgaga gtacctaaag cgaggaggac caaagggaga ggcaccaaag 780

tacgacttct tcgcaattca gtggatggag acacagaaca caggaacaaa cctattcgac 840

caggcattct cagagtcagt aattgactca ttcgacttcg acaacccaac aaagccagag 900

tggtactgca ttgagggagg aacatcacta ctagtagacg caatgaagga gacactagta 960

cacaaggtac agaacaacaa gcgagtagag gcaatttcaa ttgacctaga cgcaccagac 1020

gacggaaaca tgtcagtaaa gattggagga aaggactact caggatactc aacagtattc 1080

aacacaacag cactaggatg cctagaccga atggacctac gaggactaaa cctacaccca 1140

acacaggcag acgcaattcg atgcctacac tacgacaact caacaaaggt agcactaaag 1200

ttctcatacc catggtggat taaggactgc ggaattacat gcggaggagt agcatcaaca 1260

gacctaccac tacgaacatg cgtataccca tcatacaacc taggagacac agacgaggca 1320

gtactactag catcatacac atggtcacag gacgcaacac gaattggatc actagtaaag 1380

gacgcaccac cacagccacc aaaggaggac gagctagtag agctaattct acagaaccta 1440

gcacgactac acgcagagca catgacatac gagaagatta aggaggcata cacaggagta 1500

taccacgcat actgctgggc aaacgaccca aacgtaggag gagcattcgc actattcgga 1560

ccaggacagt tctcaaacct atacccatac ctaatgcgac cagcagcagg aggaaagttc 1620

cacattgtag gagaggcatc atcagtacac cacgcatgga ttattggatc actagagtca 1680

gcatacacag cagtatacca gttcctatac aagtacaaga tgtgggacta cctacgacta 1740

ctactagagc gatggcagta cggactacag gagctagaga caggaaagca cggaacagca 1800

cacctacagt tcattctagg atcactacca aaggagtacc aggtaaag 1848

<210>31

<211>1848

<212>DNA

<213> Artificial

<400>31

atggacaacg tagacttcgc agagtcagta cgaacacgat gggcacgacg actaatccga 60

gagaaggtag caaaggagct aaacattcta acagagcgac taggagaggt accaggaatt 120

ccaccaccac gagagggacg attcctagga ggaggatact cacacgacaa cctaccatca 180

gacccactat actcatcaat taagccagca ctactaaagg aggcaccacg agcagaggag 240

gagctaccac cacgaaaggt atgcattgta ggagcaggag tatcaggact atacattgca 300

atgattctag acgacctaaa gattccaaac ctaacatacg acattttcga gtcatcatca 360

cgaacgggag gacgactata cacacaccac ttcacagacg caaagcacga ctactacgac 420

attggagcaa tgcgataccc agacattcca tcaatgaagc gaacattcaa cctattcaag 480

cgaacaggaa tgccactaat taagtactac ctagacggag agaacacacc acagctatac 540

aacaaccact tcttcgcaaa gggagtagta gacccataca tggtatcagt agcaaacgga 600

ggaacagtac cagacgacgt agtagactca gtaggagaga agctacagca ggcattcgga 660

tactacaagg agaagctagc agagaacttc gacaagggat tcgacgagct aatgctagta 720

gacgacatga caacacgaga gtacctaaag cgaggaggac caaagggaga ggcaccaaag 780

tacgacttct tcgcaattca gtggatggag acacagaaca caggaacaaa cctattcgac 840

caggcattct cagagtcagt aattgactca ttcgacttcg acaacccaac aaagccagag 900

tggtactgca ttgagggagg aacatcacta ctagtagacg caatgaagga gacactagta 960

cacaaggtac agaacaacaa gcgagtagag gcaatttcaa ttgacctaga cgcaccagac 1020

gacggaaaca tgtcagtaaa gattggagga aaggactact caggatactc aacagtattc 1080

aacacaacag cactaggatg cctagaccga atggacctac gaggactaaa cctacaccca 1140

acacaggcag acgcaattcg atgcctacac tacgacaact caacaaaggt agcactaaag 1200

ttctcatacc catggtggat taaggactgc ggaattacat gcggaggagt agcatcaaca 1260

gacctaccac tacgaacatg cgtataccca tcatacaacc taggagacac agacgaggca 1320

gtactactag catcatacac atggtcacag gacgcaacac gaattggatc actagtaaag 1380

gacgcaccac cacagccacc aaaggaggac gagctagtag agctaattct acagaaccta 1440

gcacgactac acgcagagca catgacatac gagaagatta aggaggcata cacaggagta 1500

taccacgcat actgctgggc aaacgaccca aacgtaggag gagcattcgc actattcgga 1560

ccaggacagt tctcaaacct atacccatac ctaatgcgac cagcagcagg aggaaagttc 1620

cacattgtag gagaggcatc atcagtacac cacgcatgga ttattggatc actagagtca 1680

gcatacacag cagtatacca gttcctatac aagtacaaga tgtgggacta cctacgacta 1740

ctactagagc gatggcagta cggactacag gagctagaga caggaaagca cggaacagca 1800

cacctacagt tcattctagg atcactacca aaggagtacc aggtaaag 1848

<210>32

<211>36

<212>DNA

<213> Artificial

<400>32

cgcatatgat ggacaacgta gacttcgcag agtcag 36

<210>33

<211>36

<212>DNA

<213> Artificial

<400>33

tcagctctcg agctttacct ggtactcctt tggtag 36

<210>34

<211>39

<212>DNA

<213> Artificial

<400>34

gagtcatcat cacgaactgg aggacgacta tacacacac 39

<210>35

<211>39

<212>DNA

<213> Artificial

<400>35

gtgtgtgtat agtcgtcctc cagttcgtga tgatgactc 39

<210>36

<211>38

<212>DNA

<213> Artificial

<400>36

aattacatgc ggaggagtag catcaacaga cctaccac 38

<210>37

<211>38

<212>DNA

<213> Artificial

<400>37

gtggtaggtc tgttgatgct actcctccgc atgtaatt 38

<210>38

<211>38

<212>DNA

<213> Artificial

<400>38

aacctaggag acacagacga ggcagtacta ctagcatc 38

<210>39

<211>38

<212>DNA

<213> Artificial

<400>39

gatgctagta gtactgcctc gtctgtgtct cctaggtt 38

Claims (14)

1. A mutant characterized in that it has an amino acid sequence as shown in (a) or (b):

(a) at least one amino acid of the amino acid sequence SEQ ID NO.1 of the natural L-lysine oxidase is substituted, deleted or added, and the amino acid sequence of the mutant has more than 90 percent of homology with the amino acid sequence SEQ ID NO. 1; or

(b) At least one amino acid of the amino acid sequence SEQ ID NO.1 of the natural L-lysine oxidase is substituted, deleted or added, and the amino acid sequence of the mutant has the same function as that of the amino acid sequence SEQ ID NO. 1.

2. The mutant according to claim 1, wherein the amino acid sequence of the mutant is configured to include the amino acid sequence of one or more of the mutations in the mutation sites T122V, D229N, a417V and G438D of SEQ ID No. 1.

3. The mutant according to claim 2, wherein the amino acid sequence of the mutant is any one of SEQ ID No. 2-16.

4. A gene sequence encoding the mutant of claim 3, wherein the gene sequence is configured as any one of SEQ ID nos. 17 to 31, wherein:

the nucleic acid sequence of the mutant with the mutation site of T122V is SEQ ID NO. 17;

the nucleic acid sequence of the mutant with the mutation site of D229N is SEQ ID NO. 18;

the nucleic acid sequence of the mutant with the mutation site A417V is SEQ ID NO. 19;

the nucleic acid sequence of the mutant with the mutation site of G438D is SEQ ID NO. 20;

the nucleic acid sequence encoding the mutant with mutation sites T122V and D229N is SEQ ID No. 21;

the nucleic acid sequence encoding the mutant with the mutation sites of T122V and A417V is SEQ ID NO. 22;

the nucleic acid sequence of the mutant with the mutation sites of T122V and G438D is SEQ ID NO. 23;

the nucleic acid sequence encoding the mutant with the mutation sites of D229N and A417V is SEQ ID NO. 24;

the nucleic acid sequence of the mutant with the mutation sites of D229N and G438D is SEQ ID NO. 25;

the nucleic acid sequence of the mutant with the mutation sites of A417V and G438D is SEQ ID NO. 26;

the nucleic acid sequence of the L-lysine oxidase mutant encoding the mutants with mutation sites of T122V, D229N and A417V is SEQ ID NO. 27;

the nucleic acid sequence of the L-lysine oxidase mutant encoding the mutant with mutation sites of T122V, D229N and G438D is SEQ ID NO. 28;

the nucleic acid sequence of the mutant which codes for the mutation sites D229N, A417V and G438D is SEQ ID NO. 29;

the nucleic acid sequence of the mutant with the mutation sites of T122V, A417V and G438D is SEQ ID NO. 30;

the nucleic acid sequence encoding the mutants with mutation sites T122V, D229N, A417V and G438D is SEQ ID NO. 31.

5. The gene sequence of claim 4, wherein the nucleic acid sequence of the amplification primer pair of the mutation site T122V is SEQ ID NO.32 and SEQ ID NO. 33.

6. The gene sequence of claim 4, wherein the nucleic acid sequence of the amplification primer pair of the mutation site D229N is SEQ ID NO.34 and SEQ ID NO. 35.

7. The gene sequence of claim 4, wherein the nucleic acid sequence of the amplification primer pair of the mutation site A417V is SEQ ID NO.36 and SEQ ID NO. 37.

8. The gene sequence as claimed in claim 4, wherein the nucleic acid sequence of the amplification primer pair of mutation site G438D is SEQ ID NO.38 and SEQ ID NO. 39.

9. A method for constructing a mutant according to any one of claims 1 to 3, which comprises:

obtaining an amino acid sequence SEQ ID NO.1 of natural L-lysine oxidase in a database;

removing the repetitive sequence and the redundant sequence in the SEQ ID NO.1, and selecting an amino acid sequence with the consistency of more than 50 percent with the SEQ ID NO. 1;

carrying out multi-sequence comparison on an amino acid sequence with more than 50% of consistency with SEQ ID NO.1, and then carrying out protein three-dimensional structure prediction on the compared amino acid sequence;

and screening out mutation sites related to thermal stability.

10. A soluble protein configured to comprise a mutant according to any one of claims 1 to 3.

11. An immobilized enzyme configured to comprise the mutant according to any one of claims 1 to 3.

12. A recombinant cell configured to include the coding sequence of the mutant of any one of claims 1 to 3.

13. A recombinant vector configured to include the coding sequence of the mutant of any one of claims 1 to 3.

14. Use of the mutant according to any one of claims 1 to 3, the soluble protein according to claim 10, the immobilized enzyme according to claim 11, the recombinant cell according to claim 12 or the recombinant vector according to claim 13 for the catalytic oxidation of L-lysine.

Images