Astragaloside xylosidase mutant and application thereof
阅读说明：本技术 一种黄芪甲苷木糖苷酶突变体及其应用 (Astragaloside xylosidase mutant and application thereof ) 是由 袁其朋 程磊雨 魏斌 梁浩 刘新力 刘旭东 张伟 李静 于 2021-09-03 设计创作，主要内容包括：本发明涉及基因工程技术领域。本发明提供了一种黄芪甲苷木糖苷酶突变体及其应用,所述突变体的氨基酸序列是由SEQ ID NO：1所示的氨基酸序列突变得到；所述突变的位点包括第202位、第322位、第372和第548位中的一种或多种。本发明中的黄芪甲苷木糖苷酶突变体通过4个位点的单点突变或组合突变,能够显著提高酶水解黄芪甲苷C-(3)位置木糖苷键的水解活性,相较于野生型黄芪甲苷木糖苷酶,对底物黄芪甲苷的催化活性提高了1～20倍,大幅降低环黄芪醇工业生产的成本,使酶催化黄芪甲苷制备环黄芪醇的生产效率显著提高。(The invention relates to the technical field of genetic engineering. The invention provides an astragaloside xylosidase mutant and application thereof, wherein the amino acid sequence of the mutant is represented by SEQ ID NO: 1 is obtained by mutation of an amino acid sequence shown in the specification; the site of the mutation includes one or more of positions 202, 322, 372 and 548. The astragaloside xylosidase mutant can remarkably improve enzyme hydrolysis of astragaloside C through single-point mutation or combined mutation of 4 sites 3 Compared with wild type astragaloside xylosidase, the hydrolysis activity of the position xyloside bond is improved by 1-20 times, the industrial production cost of the cycloastragaloside is greatly reduced, and the production efficiency of preparing the cycloastragaloside by enzyme catalysis of the astragaloside is obviously improved.)
1. An astragaloside xylosidase mutant characterized in that the amino acid sequence of the mutant is represented by SEQ ID NO: 1 is obtained by mutation of an amino acid sequence shown in the specification; the site of the mutation includes one or more of positions 202, 322, 372 and 548.
2. The astragaloside xylosidase mutant according to claim 1, wherein the mutation at position 202 is: tryptophan is mutated to glycine, alanine, aspartic acid, glutamic acid, threonine or serine.
3. The astragaloside xylosidase mutant according to claim 2, wherein the mutation at position 322 is: histidine was mutated to glycine or alanine.
4. The astragaloside xylosidase mutant according to claim 3, wherein the 372 nd mutation is: tyrosine is mutated into glycine, alanine, aspartic acid or glutamic acid.
5. The astragaloside xylosidase mutant according to any one of claims 1-4, wherein the 548 th mutation is: tyrosine is mutated into glycine, alanine, aspartic acid or glutamic acid.
6. A recombinant plasmid comprising the mutant nucleotide sequence of any one of claims 1 to 5.
7. A host cell comprising the recombinant plasmid of claim 6.
8. Use of the mutant of any one of claims 1-5 in catalyzing the production of cycloastragenol from astragaloside IV.
9. The use according to claim 8, wherein the mutant catalyzes the hydrolysis of C of astragaloside IV3Positional xylosidic bonds.
Technical Field
The invention relates to the technical field of genetic engineering, in particular to an astragaloside xylosidase mutant and application thereof.
Background
Cycloastragenol is a tetracyclic triterpene compound with molecular formula of C30H50O5(490.71) it is a colorless needle-like crystal. Researches show that the cycloastragenol is an effective telomerase activator, and can prolong telomeres by activating telomerase, so that the effects of resisting aging and improving the cell proliferation capacity are achieved. With the increasing severity of the global aging problem, modern agricultural development, productivity supply and health care and security systems are all subjected to significant impact and impact. Therefore, the high-efficiency preparation of the cycloastragenol becomes a research hotspot.
Cycloastragenol exists mainly in the form of astragaloside IV in natural plants, and the content of free cycloastragenol is very low. Removing astragaloside IV by hydrolysis3Positional xylose and C6The glucose at the position can realize the production of the cycloastragenol.
The traditional acid hydrolysis method for preparing cycloastragenol by using astragaloside IV generates a great amount of highly polluted wastewater, and moreover, a byproduct of astragenol is generated under the conditions of strong acid and high temperature, the byproduct of astragenol is difficult to separate from the product cycloastragenol, and the resource utilization rate is low. Although the generation of the astraganol can be effectively avoided by adding the redox step, the steps are complex, and the industrial application prospect is not sufficient. The method for preparing cycloastragenol by hydrolyzing astragaloside IV through an enzyme catalysis method has the advantages of high catalysis efficiency, high specificity, mild reaction conditions, environmental friendliness and the like, but the activity of the existing astragaloside IV xylosidase bond hydrolase is limited. How to improve the performance of the enzyme and thus the production efficiency of the cycloastragenol is a problem to be solved urgently.
Disclosure of Invention
The invention aims to provide an astragaloside xylosidase mutant and application thereof, which can reduce the cost of industrial production of cycloastragenol and obviously improve the production efficiency of preparing the cycloastragenol by catalyzing the enzyme astragaloside.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an astragaloside xylosidase mutant, the amino acid sequence of which is represented by SEQ ID NO: 1 is obtained by mutation of an amino acid sequence shown in the specification; the site of the mutation includes one or more of positions 202, 322, 372 and 548.
Preferably, the mutation at position 202 is: tryptophan is mutated to glycine, alanine, aspartic acid, glutamic acid, threonine or serine.
Preferably, the mutation at position 322 is: histidine was mutated to glycine or alanine.
Preferably, the 372 nd mutation is: tyrosine is mutated into glycine, alanine, aspartic acid or glutamic acid.
Preferably, the 548 th mutation is: tyrosine is mutated into glycine, alanine, aspartic acid or glutamic acid.
The invention also provides a recombinant plasmid containing the nucleotide sequence of the astragaloside xylosidase mutant.
The invention also provides a host cell containing the recombinant plasmid.
The invention also provides application of the mutant in catalyzing astragaloside to produce cycloastragenol.
Preferably, the mutant catalyzes astragaloside IVRelieve it C3Positional xylosidic bonds.
The invention provides an astragaloside xylosidase mutant and application thereof, wherein the amino acid sequence of the mutant is represented by SEQ ID NO: 1 is obtained by mutation of an amino acid sequence shown in the specification; the site of the mutation includes one or more of positions 202, 322, 372 and 548. The astragaloside xylosidase mutant can remarkably improve enzyme hydrolysis of astragaloside C through single-point mutation or combined mutation of 4 sites3Compared with wild type astragaloside xylosidase, the hydrolysis activity of the position xyloside bond is improved by 1-20 times, the industrial production cost of the cycloastragaloside is greatly reduced, and the production efficiency of preparing the cycloastragaloside by enzyme catalysis of the astragaloside is obviously improved.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
EXAMPLE 1 determination of mutation sites
Conservative analysis of the astragaloside xylosidase amino acid sequence (SEQ ID NO: 1) was performed using WebLogi 3 online software, where the set of homologous sequence data for the conservative analysis was exported by the Consensuss Finder software. The three-dimensional MODEL of the astragaloside xylosidase is established by a SWISS-MODEL online server (https:// swissmodel.expasy.org/interactive), and the used template is PDB ID: 6Q 7I. The quality of the constructed model is evaluated by using software such as ProSA and PROCHECK. The constructed astragaloside IV xylosidase three-dimensional structure was structurally superimposed and visualized with template (6Q7I) using Pymol software. The results show that Asp407 and Glu611 in astragaloside xylosidase have similar position and orientation to the template catalytic residues Asp307 (nucleophile) and Glu509 (acidifying or alkalizing agent). Furthermore, sequence conservation analysis shows that Asp407 and Glu611 are highly conserved through evolution. Therefore, Asp407 (nucleophile) and Glu611 (acidifying or alkalizing agent) are predicted to be the catalytic residues of astragaloside xylosidase. Further, carrying out molecular docking calculation on the astragaloside xylosidase model and a substrate astragaloside. The three-dimensional structure of astragaloside is obtained from a PubChem (https:// PubChem. ncbi. nlm. nih. gov /) library, and energy minimization is carried out in an MMFF94 force field. Asp407 and Glu611 in the enzyme Astragaloside xylosidase define the docking pocket, and the DOCK 6.9 program was run on the YInfo cloud platform (http:// closed. yinfotek. com /) for semi-flexible docking calculations of substrate and enzyme, and the energy of the output docking conformation was scored using a gridding scoring function. The results show that Asn131, Trp202, H322, Arg336, Try372, Asn547 and Tyr548 in astragaloside xylosidase have interaction with astragaloside, mainly hydrogen bond or hydrophobic interaction, so that the 7 sites are determined as the hot spot residues of mutation.
Example 2 primer design
As set forth in SEQ ID NO: 2 as template, designing primer sequences for site-directed mutagenesis (N131A, N131G, N131S, W202G, W202A, W202D, W202E, W202T, W202S, H322A, H322G, R336G, R336A, Y372G, Y372A, Y372D, Y372E, N547A, N547G, Y548G, Y548A, Y548D, Y548E, Y548V and Y548T), wherein specific primer sequences are shown in Table 1:
TABLE 1
Example 3 obtaining of DNA sequence of Astragaloside xylosidase mutant
Obtaining a mutant nucleotide sequence by utilizing a site-directed mutagenesis technology, and introducing a mutation site on the astragaloside IV xylosidase in a PCR amplification mode. Taking the nucleotide sequence SEQ ID NO: 2 as a template, amplifying a partial DNA fragment by using F-EcoRI and a downstream primer of a mutation site, amplifying a partial DNA fragment by using R-NotI and an upstream primer of the mutation site, and completing the first round of PCR amplification. And then mixing the DNA fragments obtained after the two times of amplification and carrying out a second round of PCR reaction so as to obtain a complete mutated DNA sequence. The PCR amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 2min, annealing at 58 ℃ and extension at 72 ℃ for 180s for 30 cycles, and final extension at 72 ℃ for 10 min. The PCR product was subjected to 1% agarose gel electrophoresis and then gel-cut and recovered. The first round of PCR amplification system is shown in Table 2, and the second round of PCR amplification system is shown in Table 3.
TABLE 2
TABLE 3
Example 4 plasmid construction
After the PCR reaction is finished, separating, purifying and recycling the PCR product by using an OMEGA gel recycling kit. The recovered DNA fragment and the pPIC9k vector were subjected to double digestion with EcoRI and NotI restriction enzymes, respectively, under the reaction conditions shown in Table 4.
TABLE 4
The digested product was then recovered and purified, and recombinant vectors (pPIC 9-N131, pPIC 9-W202, pPIC 9-H322, pPIC 9-R336, pPIC 9-Y372, pPIC 9-N547, pPIC 9-Y548) were obtained by ligating the digested gene sequences with the digested vector, respectively, using T ligase. The ligation system is shown in Table 5.
TABLE 5
Example 5 construction of Pichia pastoris recombinant hosts
The recombinant vectors obtained above (pPIC 9-N131, pPIC 9-W202, pPIC 9-H322, pPIC 9-R336, pPIC 9-Y372, pPIC 9-N547, pPIC 9-Y548) were subjected to single linearization using SalI restriction endonuclease; then the linearized recombinant vectors are respectively transformed into an activated Pichia pastoris GS115 to obtain enzyme-producing host cells (GS115-N131A, GS115-N131G, GS115-N131S, GS115-W202G, GS115-W202A, GS115-W202D, GS115-W202E, GS115-W202T, GS115-W202S, GS115-H322A, GS115-H322G, GS115-R G, GS115-R336A, GS115-Y372G, GS115-Y372A, GS115-Y372D, GS115-Y372E, GS115-N547A, GS115-N547G, GS115-Y G, GS115-Y548A, GS115-Y D, GS115-Y548, GS 115-Y36548 548 and GS115-Y T).
Example 6 expression and purification of Astragaloside xylosidase mutants
Inoculating the obtained host cell for producing enzyme into BMMG culture medium, and culturing at 30 deg.C and 220rpm for 12 hr; then, the cells were collected and washed 2 times with sterile water, and inoculated into BMMY medium and cultured at 30 ℃ at 220rpm, and induced by adding 1% methanol every 24 hours. After culturing for 4 days, the cells were removed by centrifugation and filtration to obtain a fermentation broth, and the fermentation broth was purified using a nickel column to finally obtain a purified astragaloside xylosidase mutant (N131A, N131G, N131S, W202G, W202A, W202D, W202E, W202T, W202S, H322A, H322G, R336G, R336A, Y372G, Y372A, Y372D, Y372E, N547A, N547G, Y548G, Y548A, Y548D, Y548E, Y548V, Y548T).
Example 7 Studies on enzyme Activity of Astragaloside xylosidase mutant
And (3) measuring enzyme activity: the substrate is 5mM astragaloside, the astragaloside xylosidase mutant is added, the reaction is carried out for 1h under the conditions of pH 5.5 and temperature 45 ℃, a proper amount of methanol is added for dissolution after the reaction is finished, the content of the astragaloside is detected by high performance liquid chromatography, the enzyme activity is calculated, and the result is shown in Table 6. Wherein the enzyme activity of the astragaloside xylosidase mutant is defined as the enzyme amount required for removing 1 mu mol of astragaloside per minute, and the specific enzyme activity is the enzyme activity of the unit mg of protein.
TABLE 6
+ represents that the enzyme activity is improved by 1-5 times relative to the wild type, + + represents that the enzyme activity is improved by 5-10 times relative to the wild type, and, + + + represents that the enzyme activity is improved by 10-20 times relative to the wild type, and-represents that the enzyme activity is not obviously changed or reduced relative to the wild type.
The results of combining the single mutants with significantly improved enzyme activity are shown in Table 7.
TABLE 7
Enzyme
Specific enzyme activity
WT
-
W202G/H322G
+++
W202A/H322A
+++
W202D/Y372G
++
W202E/Y548G
++
H322G/Y372A
+++
Y372G/Y548G
+++
Y372A/Y548G
++
+ represents that the enzyme activity is improved by 1-5 times relative to the wild type, + + represents that the enzyme activity is improved by 5-10 times relative to the wild type, and, + + + represents that the enzyme activity is improved by 10-20 times relative to the wild type, and-represents that the enzyme activity is not obviously changed or reduced relative to the wild type.
Example 8 application of Astragaloside xylosidase mutant W202G in preparation of cycloastragenol by converting Astragaloside IV
100mg of 10% by mass of Astragaloside powder was added to 10mL of 100 mM (pH 6.0) phosphate buffer, and then 0.1mg of Astragaloside xylosidase mutant W202G and 0.1mg of glucosidase were added, followed by a complete reaction at 45 ℃ at 200r/min for 3 hours. Liquid phase detection after the reaction is finished shows that the yield of the cycloastragenol is as high as 98.2 percent.
Example 9 application of Astragaloside xylosidase mutant H322G in preparation of cycloastragenol by converting Astragaloside IV
100mg of 10% by mass of Astragaloside powder was added to 10mL of 100 mM (pH 6.0) phosphate buffer, and then 0.2mg of Astragaloside xylosidase mutant H322G and 0.1mg of glucosidase were added, followed by a complete reaction at 45 ℃ at 200r/min for 3 hours. After the reaction is finished, liquid phase detection shows that the yield of the cycloastragenol is as high as 98.6 percent.
Example 10 application of Astragaloside xylosidase mutant Y372E in preparation of cycloastragenol by converting Astragaloside IV
100mg of 10% by mass of Astragaloside powder was added to 10mL of 100 mM (pH 6.0) phosphate buffer, and then 0.2mg of Astragaloside xylosidase mutant Y372E and 0.1mg of glucosidase were added, followed by a complete reaction at 45 ℃ at 200r/min for 3 hours. After the reaction is finished, liquid phase detection shows that the yield of the cycloastragenol is as high as 98.7 percent.
Example 11 application of the Astragaloside xylosidase mutant Y548G in the preparation of cycloastragenol by converting Astragaloside IV
100mg of 10% by mass of Astragaloside powder was added to 10mL of 100 mM (pH 6.0) phosphate buffer, and then 0.2mg of Astragaloside xylosidase mutant Y548G and 0.1mg of glucosidase were added, followed by a complete reaction at 45 ℃ at 200r/min for 3 hours. After the reaction is finished, liquid phase detection shows that the yield of the cycloastragenol is as high as 98.1 percent.
Example 12 application of Astragaloside xylosidase mutant H322G/Y372A in preparation of cycloastragenol by converting Astragaloside IV
100mg of 10% by mass of Astragaloside powder was added to 10mL of 100 mM (pH 6.0) phosphate buffer, and then 0.1mg of Astragaloside xylosidase mutant H322G/Y372A and 0.1mg of glucosidase were added thereto, followed by a complete reaction at 45 ℃ at 200r/min for 3 hours. Liquid phase detection after the reaction is finished shows that the yield of the cycloastragenol is as high as 98.3 percent.
From the above embodiments, the present invention provides an astragaloside xylosidase mutant and an application thereof, wherein the amino acid sequence of the mutant is represented by SEQ ID NO: 1 is obtained by mutation of an amino acid sequence shown in the specification; the site of the mutation includes one or more of positions 202, 322, 372 and 548. Astragaloside IV wood in the inventionThe glycosidase mutant can remarkably improve enzyme hydrolysis of astragaloside IV C through single-point mutation or combined mutation of 4 sites3Compared with wild type astragaloside xylosidase, the hydrolysis activity of the position xyloside bond is improved by 1-20 times, the industrial production cost of the cycloastragaloside is greatly reduced, and the production efficiency of preparing the cycloastragaloside by enzyme catalysis of the astragaloside is obviously improved.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Lilium Viridium Biotechnology Ltd
Beijing university of chemical industry
<120> astragaloside xylosidase mutant and application thereof
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<213> Artificial Sequence (Artificial Sequence)
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Met Ser Lys Phe Leu Ser Trp Ser Val Leu Leu Gln Gly Val Ser Tyr
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Thr Leu Ala Ala Pro Gln Gln Gly Lys Cys Leu Pro Ala Val Asn Val
20 25 30
Gly Tyr Ala Ala Asn Ile Thr Phe Val Gly Cys Tyr Thr Asp Asn Ser
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Thr Arg Ile Leu Gln Gly Gly Ser Thr Val Pro Pro Asn Gly Asn Asp
50 55 60
Pro Gln Ser Cys Ala Asp Ser Cys Gly Ala Ser Gly Phe Thr Tyr Ala
65 70 75 80
Gly Val Glu Tyr Gly Ser Gln Cys Tyr Cys Gly Ser Thr Ile Leu Ser
85 90 95
Gly Ala Gln Gln Gln Ala Asp Ser Ala Cys Thr Met Thr Cys Ser Gly
100 105 110
Asn Ser Ser Glu Ile Cys Gly Gly Thr Trp Leu Val Asp Ile Tyr Gln
115 120 125
Ile Ser Asn Pro Ser Ser Thr Pro Val Pro Leu Ser Gly Ala Val Lys
130 135 140
Pro Asn Cys Thr Ala Asp Pro Leu Cys Ser Asn Pro Val Cys Asn Ala
145 150 155 160
Ala Leu Asp Pro Leu Thr Arg Ala Lys Gly Leu Val Asp Ala Leu Thr
165 170 175
Phe Asp Glu Lys Ile Gln Asn Thr Gln Asn Gly Ser Pro Gly Ser Ala
180 185 190
Arg Leu Gly Leu Pro Gly Tyr Gln Trp Trp Ser Glu Ala Leu His Gly
195 200 205
Val Ala Ser Ser Pro Gly Val Thr Phe Gln Thr Gly Asn Phe Ser Tyr
210 215 220
Ala Thr Ser Phe Pro Gln Pro Ile Leu Met Ser Ala Ala Phe Asp Asp
225 230 235 240
Ala Leu Ile Gln Gln Val Gly Thr Val Val Ser Ile Glu Gly Arg Ala
245 250 255
Phe Ser Asn Tyr Gly Asn Ala Gly Leu Asp Phe Trp Thr Pro Asn Ile
260 265 270
Asn Pro Phe Arg Asp Pro Arg Trp Gly Arg Gly Gln Glu Thr Pro Gly
275 280 285
Glu Asp Pro Phe His Ile Ala Arg Tyr Val Tyr Asn Leu Val Asp Gly
290 295 300
Leu Gln Asn Gly Ile Gly Pro Thr Asn Pro Arg Val Val Ala Thr Cys
305 310 315 320
Lys His Phe Ala Gly Tyr Asp Ile Glu Asp Trp Glu Gly Asn Ala Arg
325 330 335
Tyr Gly Phe Asn Ala Ile Ile Ser Thr Gln Asp Leu Ser Glu Tyr Tyr
340 345 350
Leu Pro Pro Phe Lys Ser Cys Ala Arg Asp Ala Lys Val Asp Ala Ile
355 360 365
Met Cys Ser Tyr Asn Ala Val Asn Gly Ile Pro Thr Cys Ala Asp Ser
370 375 380
Tyr Leu Leu Asp Thr Ile Leu Arg Asp His Trp Asn Trp Asn Gln Thr
385 390 395 400
Gly Arg Trp Val Thr Ser Asp Cys Asp Ala Ile Gly Asn Ile Phe Thr
405 410 415
Asp His His Tyr Thr Ser Thr Ala Ala Ala Ala Ala Ala Asp Ala Leu
420 425 430
Asn Ala Gly Thr Asn Leu Asp Cys Gly Thr Thr Met Ser Asn Asn Leu
435 440 445
Ala Ala Ala Ser Ala Gln Asp Leu Phe Gln Asn Ala Thr Leu Asp Thr
450 455 460
Ala Leu Thr Tyr Leu Tyr Ser Ser Leu Val Arg Leu Gly Trp Phe Asp
465 470 475 480
Glu Glu Thr Ser Pro Tyr Arg Ser Leu Asp Trp Ser Asp Val Gly Thr
485 490 495
Pro Ala Ser Gln Gln Leu Ala Ile Arg Ala Ala Val Glu Gly Ile Val
500 505 510
Leu Leu Lys Asn Asp Lys Thr Lys Val Leu Pro Leu Ser Ser His Arg
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Gln Thr Ile Ala Leu Ile Gly Pro Tyr Ala Asn Ala Thr Thr Gln Leu
530 535 540
Gln Gly Asn Tyr Ala Gly Val Ala Glu Tyr Ile Arg Thr Leu Val Trp
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Gly Ala Glu Gln Ala Gly Tyr Asn Val Glu Tyr Ala Leu Gly Thr Asp
565 570 575
Ile Asn Ser Thr Asp Thr Ser Gly Phe Ser Ala Ala Val Ala Ala Ala
580 585 590
Asn Ala Ser Asp Ile Ile Ile Tyr Ala Gly Gly Ile Asp Asn Ser Ile
595 600 605
Glu Ala Glu Ala Met Asp Arg Asp Thr Ile Ser Trp Pro Gly Asn Gln
610 615 620
Leu Gln Leu Val Asp Glu Leu Ser Gln Val Gly Lys Pro Leu Ile Val
625 630 635 640
Leu Gln Phe Gly Gly Gly Gln Leu Asp Asp Ser Ala Leu Leu Glu Asn
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Glu Lys Val Asn Ala Ile Leu Trp Ala Gly Tyr Pro Ser Gln Ala Gly
660 665 670
Gly Gln Ala Val Phe Asp Ile Leu Thr Gly Lys Ser Ala Pro Ala Gly
675 680 685
Arg Leu Pro Ile Thr Gln Tyr Pro Ala Asn Tyr Thr Asn Glu Ile Pro
690 695 700
Met Thr Asp Met Ala Leu Arg Pro Asn Gly Thr Asn Pro Gly Arg Thr
705 710 715 720
Tyr Arg Trp Tyr Asp Asp Ala Val Ile Pro Phe Gly Tyr Gly Leu His
725 730 735
Tyr Thr Ser Phe Asp Val Ser Trp Ala Ser Lys Lys Leu Gly Pro Tyr
740 745 750
Asn Thr Ala Ser Leu Gly His Val Ser Lys Ser Gln Tyr Pro Asp Thr
755 760 765
Ala Ala Phe Asp Thr Phe His Ile Asp Val Lys Asn Thr Gly Lys Val
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Thr Ser Asp Tyr Val Ala Leu Leu Phe Ala Ser Thr Lys Asn Ala Gly
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Pro Ala Pro Tyr Pro Ile Lys Thr Leu Val Gly Tyr Ala Arg Ala Pro
805 810 815
Ser Ile Lys Pro Gly Glu Thr Arg Ser Val Ser Leu Asp Val Thr Leu
820 825 830
Gly Ala Ile Ala Arg Thr Ala Glu Asn Gly Asp Leu Val Leu Tyr Pro
835 840 845
Gly Thr Tyr Thr Leu Glu Val Asp Val Gly Gln His Tyr Pro Thr Ala
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Glu Phe Gln Val Asn Gly Pro Asp Lys Val Leu Asp Ser Phe Pro Gln
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Pro Pro Ser Ser
<210> 2
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<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgtctaaat ttttgtcttg gtctgttttg ttgcaaggtg tttcttatac tttggctgcc 60
ccacaacaag gtaaatgttt gcctgctgtt aacgttggat acgctgctaa cattactttt 120
gttggttgtt acactgacaa ctccactcgt attttgcaag gtggttccac tgttccacca 180
aacggtaacg acccacaatc ttgtgctgac tcctgtggtg cttctggttt cacctacgcc 240
ggtgttgagt acggttccca atgttactgt ggttctacca ttctgtcagg tgctcagcaa 300
caagccgatt ctgcttgtac catgacttgt tccggtaatt cttctgagat ttgtggtggt 360
acttggttgg ttgatatcta ccagatctct aacccttctt ctactccagt tccattgtct 420
ggtgctgtta agcctaattg tactgctgat cctttgtgtt ctaaccccgt ttgtaacgct 480
gcactggatc cattgactag agctaaggga ttagttgacg ctttgacttt tgacgaaaag 540
attcaaaaca cccagaacgg aagtcctggt tccgctcgtt tgggtttgcc aggttatcag 600
tggtggtctg aagctttgca cggagttgct tcttctccag gtgttacttt ccaaaccggt 660
aacttttcct acgctacttc tttcccacaa ccaattctta tgtccgctgc ttttgatgat 720
gctctgattc aacaagttgg tactgttgtt tcaattgaag gtagagcttt ctccaactac 780
ggtaacgccg gactggattt ttggactcct aacattaacc ctttccgtga tcctagatgg 840
ggtagaggtc aagaaactcc tggtgaggac cctttccaca ttgctcgtta cgtttacaat 900
ctggttgatg gtctgcaaaa cggtatcggt cctactaacc ctagagttgt tgctacctgt 960
aagcactttg ctggttacga cattgaggat tgggaaggta acgctagata cggttttaac 1020
gctattattt caactcaaga tttgtccgag tactacctgc ctccttttaa atcttgtgct 1080
agagacgcta aggttgacgc tattatgtgt tcttataatg ctgttaacgg tattcctacc 1140
tgtgctgact cctacttgtt ggacaccatt ttgagagatc attggaactg gaaccaaacc 1200
ggtagatggg tgacctctga ttgtgacgct attggtaata tcttcactga tcaccactac 1260
acttccactg ctgctgctgc tgccgcagat gctttgaatg ccggtaccaa cttggactgt 1320
ggtaccacca tgtctaacaa tcttgctgct gcttctgccc aggatctgtt ccaaaacgcc 1380
actctagaca ccgccttgac ttatttgtac tcgtctttgg ttcgtttggg ttggtttgac 1440
gaagaaacct ctccataccg atctttggat tggtctgacg tcggtactcc agcttcccag 1500
caactggcca tcagagccgc tgttgaaggt atcgttttgt tgaagaacga caaaactaag 1560
gttttgcctt tgtcatctca ccgtcaaacc atcgctctta ttggaccata cgctaacgct 1620
accactcaat tgcaaggtaa ctacgctggt gtcgctgaat acatcagaac tttggtttgg 1680
ggtgctgagc aagctggtta taacgttgaa tatgctttgg gtactgatat taattcaact 1740
gatacttctg gtttctctgc cgctgtcgca gctgctaacg cttctgatat catcatctac 1800
gctggtggta ttgataactc aatcgaggca gaggctatgg acagagatac tatctcttgg 1860
ccaggtaatc aattgcaatt ggttgatgaa ctttctcaag tcggtaaacc tttaattgtt 1920
cttcaattcg gaggtggaca acttgatgac tctgccttgt tggaaaatga gaaggttaac 1980
gctattcttt gggctggtta cccatcccaa gctggtggtc aagccgtttt tgatatcttg 2040
actggtaaat ccgctcctgc tggtagactg cctattaccc aataccctgc taactacacc 2100
aacgaaatcc caatgactga catggctttg agaccaaatg gtactaaccc aggtagaacc 2160
tatagatggt acgatgatgc tgtgattcct ttcggttacg gtttgcatta cactagtttc 2220
gacgtttctt gggcttccaa gaagttgggt ccatacaaca ccgcctcact gggtcacgtt 2280
tctaagtctc aataccctga taccgctgcc tttgacactt ttcatatcga cgtcaagaac 2340
actggtaaag ttactagtga ttacgtcgct ctgctgttcg cttctactaa gaacgctggt 2400
ccagcccctt accctatcaa gaccttggtc ggttatgcta gagccccatc aatcaagcca 2460
ggtgaaacta gatccgtctc cttggatgtt actttgggtg ctattgctag aactgctgaa 2520
aacggtgacc tggtcttgta cccaggaact tacaccttgg aggttgacgt cggtcaacac 2580
tacccaactg ctgagttcca ggtcaacggt cctgataagg tcttggattc tttcccacaa 2640
cctccatctt ct 2652
<210> 3
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gggaaagaat tcgccccaca acaagg 26
<210> 4
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gggaaagcgg ccgcgtgatg gtggtggtga tg 32
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ccagatctct gctccttctt c 21
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ggtctagaga cgaggaagaa g 21
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ccagatctct ggcccttctt c 21
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ggtctagaga ccgggaagaa g 21
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ccagatctct agcccttctt c 21
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ggtctagaga tcgggaagaa g 21
<210> 11
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
atcagtgggg ctctgaagc 19
<210> 12
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tagtcacccc gagacttcg 19
<210> 13
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
atcagtgggc ttctgaagc 19
<210> 14
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
tagtcacccg aagacttcg 19
<210> 15
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
atcagtggga ctctgaagc 19
<210> 16
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
tagtcaccct gagacttcg 19
<210> 17
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
atcagtggga gtctgaagc 19
<210> 18
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
tagtcaccct cagacttcg 19
<210> 19
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
atcagtggac gtctgaagc 19
<210> 20
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
tagtcacctg cagacttcg 19
<210> 21
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
atcagtggag ctctgaagc 19
<210> 22
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
tagtcacctc gagacttcg 19
<210> 23
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
acctgtaagg cttttgctg 19
<210> 24
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
tggacattcc gaaaacgac 19
<210> 25
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
acctgtaagg gctttgctg 19
<210> 26
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
tggacattcc cgaaacgac 19
<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ggtaacgctg gctacggttt 20
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ccattgcgac cgatgccaaa 20
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
ggtaacgctg cttacggttt 20
<210> 30
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
ccattgcgac gaatgccaaa 20
<210> 31
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
gtgttctggc aatgctgtta acgg 24
<210> 32
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
cacaagaccg ttcgacaatt gcc 23
<210> 33
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
gtgttctgct aatgctgtta acgg 24
<210> 34
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
cacaagacga ttcgacaatt gcc 23
<210> 35
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gtgttctgac aatgctgtta acgg 24
<210> 36
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
cacaagactg ttcgacaatt gcc 23
<210> 37
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gtgttctgag aatgctgtta acgg 24
<210> 38
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
cacaagactc ttcgacaatt gcc 23
<210> 39
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
gcaaggtgct tacgctggt 19
<210> 40
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
cgttccacga atgcgacca 19
<210> 41
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
gcaaggtggc tacgctggt 19
<210> 42
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
cgttccaccg atgcgacca 19
<210> 43
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
caaggtaacg gcgctggtgt c 21
<210> 44
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
gttccattgc cgcgaccaca g 21
<210> 45
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
caaggtaacg ctgctggtgt c 21
<210> 46
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
gttccattgc gacgaccaca g 21
<210> 47
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
caaggtaacg acgctggtgt c 21
<210> 48
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
gttccattgc tgcgaccaca g 21
<210> 49
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
caaggtaacg aggctggtgt c 21
<210> 50
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
gttccattgc tccgaccaca g 21
<210> 51
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
caaggtaacg tcgctggtgt c 21
<210> 52
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
gttccattgc agcgaccaca g 21
<210> 53
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
caaggtaaca ctgctggtgt c 21
<210> 54
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
gttccattgt gacgaccaca g 21
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