Example 4: study of enzymatic Properties of pullulanase of the present invention
The method comprises the following specific steps:
(1) the optimum pH and pH stability of the pullulanase of the invention
Preparing phosphate buffer solutions with pH values of 4.0, 5.0, 6.0, 7.0 and 8.0 respectively to replace buffer solutions in the pullulanase activity determination method, determining the pullulanase activity at 50 ℃, and calculating relative enzyme activity by taking the highest enzyme activity as 100% and the other enzyme activities as comparison to investigate the optimum action pH of the enzyme (the detection result is shown in figure 2);
preparing phosphate buffer solutions with pH values of 4.0, 5.0, 6.0, 7.0 and 8.0 respectively to replace the buffer solution in the pullulanase activity determination method, storing the purified enzymes at 25 ℃ for 24 hours respectively in the buffer system, determining the pullulanase activity at 50 ℃, and calculating the residual enzyme activity by comparing the enzyme activity after storage with the initial enzyme activity of 100% to examine the pH stability (the detection result is shown in figure 3).
As can be seen from FIG. 2, the optimum pH of the pullulanase of the present invention is 6.5, the enzyme activity retention rate at pH 7.0 is 91%, and when the pH is lower than 6.5 or higher than 7.0, the pullulanase activity is rapidly decreased, i.e., the pullulanase of the present invention has good catalytic activity in a weakly acidic environment, but has weak catalytic activity in neutral and weakly alkaline environments, and after deviating from this range, the enzyme activity is rapidly decreased and almost no enzyme activity is present, which indicates that the optimum pH of the pullulanase of the present invention is mild, but the range is narrow.
As can be seen from FIG. 3, the pullulanase of the present invention has good pH stability under the weak acidic to neutral condition of pH 5.5-7.5, the preservation rate of the enzyme activity is above 80%, the residual enzyme activity is very low below pH 5.0, and the preservation rate of the enzyme activity begins to decrease after the pH is above 7.5, which indicates that the pullulanase of the present invention is suitable for preservation under the neutral or weak acidic condition. .
(2) The optimum temperature and the temperature stability of the pullulanase of the invention
Respectively measuring pullulanase enzyme activity at 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, and 60 deg.C, calculating relative enzyme activity by taking the highest enzyme activity as 100%, and comparing the other enzyme activities with the highest enzyme activity, to examine the optimum action temperature of enzyme (see figure 4);
and (3) respectively preserving the purified enzyme at 30 ℃ and 40 ℃ for 12h, taking out part of enzyme liquid after a period of time, quickly cooling, measuring the pullulanase activity, and calculating the residual enzyme activity by taking the initial enzyme activity as 100% and comparing the enzyme activity with the enzyme activity after heat preservation so as to investigate the temperature stability of the pullulanase (the detection result is shown in figure 5).
As can be seen from FIG. 4, the optimum temperature of the pullulanase of the present invention is 50 ℃, more than 90% of the activity is maintained between 45 ℃ and 50 ℃, and the enzyme activity rapidly decreases when the temperature is higher than 50 ℃, which indicates that the pullulanase of the present invention has mild action conditions and is very suitable for the catalytic process under the condition of moderate temperature.
As can be seen from FIG. 5, the enzyme activity of the pullulanase of the present invention decreased rapidly in the first 20 hours at 30 ℃ and 40 ℃, and the enzyme activity remained about 50% after 30 hours of heat preservation at 40 ℃; and at the temperature of 30 ℃, the enzyme activity at the early stage is reduced quickly, but the enzyme activity at the later stage is kept stable, and the half-life period can reach more than 90 h.
(3) Effect of Metal chelators and Metal ions on the pullulanase of the present invention
1mL of a 1.0% pullulan solution (substrate) and 0.9mL of 50 mmol.L-1pH 6.5 phosphate buffer into a test tube, and metal ion Ca2+、Fe2+、Zn2+、Mg2+、Zn2+、Cu2+、Co2+And adding the metal chelating agent EDTA mother liquor into the test tube respectively to make the metal ions Ca2+、Fe2+、Zn2+、Mg2+、Zn2+、Cu2+、Co2+And the final concentration of the metal chelating agent EDTA mother liquor in the test tube is 1mM and 5mM respectively to obtain a mixed solution; preheating the mixed solution in a 50 ℃ water bath for about 10min, then adding 0.1mL of diluted enzyme solution sample into the mixed solution, shaking and uniformly mixing, incubating at 50 ℃ for 10min for reaction, quickly adding 3mL of DNS (Domain name System) to terminate the reaction after the reaction is finished, developing in a boiling water bath for 7min, cooling in ice water, finally adding 10mL of distilled water into the reaction system, uniformly mixing, and measuring the light absorption value at 540 nm; the enzyme activity measured by using a sample (Control group) without adding metal ions and metal chelating agents is 100%, and the ratio of the enzyme activity measured by using other substrates to the activity is the relative enzyme activity (the result is shown in table 1).
As is clear from Table 1, 1 mmol. L-1The EDTA has strong inhibition effect on the pullulanase, almost inactivates the pullulanase, and the residual enzyme activity is 4.8 percent; 5 mmol. L-1After the EDTA is used for treating the pullulanase, the residual enzyme activity is 3.2 percent, which shows that the pullulanase needs the participation of metal ions when playing a role;
and in 1 mmol. L-1In the metal ion of (2), Mn2+、Co2+The pullulanase has moderate inhibition effect on the pullulanase; fe2+、Zn2+、Cu2+The inhibition effect on the pullulanase is very obvious; ca2+The pullulanase has a certain promotion effect on the pullulanase; at 5 mmol. L-1All of the metal ions of (a) have no promoting effect on the pullulanase of the present invention, wherein a high concentration of Mn2+The inhibitory effect of the ion on the enzyme is minimal, next to Ca2+The inhibition of the enzyme by ions, other metal ions at high concentrations, is evident.
TABLE 1 Effect of Metal ions and Metal chelators on enzyme Activity
Metal ion or chelating agent (1mM)
|
Relative activity* |
Metal ion or chelating agent (5mM)
|
Relative activity
|
Control
|
100.0±2.2
|
Control
|
100.0±3.97
|
Ca2+ |
107.50±3.2
|
Ca2+ |
93.35±3.66
|
Fe2+ |
17.45±0.52
|
Fe2+ |
6.62±0.33
|
Zn2+ |
12.01±0.36
|
Zn2+ |
6.54±0.32
|
Mg2+ |
93.20±2.8
|
Mg2+ |
95.97±2.79
|
Mn2+ |
72.35±2.2
|
Mn2+ |
71.62±3.58
|
Cu2+ |
1.40±0.14
|
Cu2+ |
0.94±0.04
|
Co2+ |
52.85±1.59
|
Co2+ |
19.71±0.98
|
EDTA
|
4.75±0.14
|
EDTA
|
3.17±0.98 |
(4) Kinetic parameters of the pullulanase of the present invention
Respectively taking pullulan solutions with the concentrations of 0.1, 0.25, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0 and 10.0mg/mL as substrates, measuring the initial hydrolysis activity of the pullulanase at the temperature of 50 ℃ and the pH value of 6.5, and fitting the data by adopting a nonlinear regression method in GraphPad Prism 7.0 software to respectively obtain the K of the Michaelis-Menten equationmAnd VmaxValue, then K is calculatedcatAnd Kcat/KmA value;
Kcatthe calculation formula of the value is: kcat=Vmax/M/106124.74/60; wherein M is the mass of the enzyme added in the reaction, and the unit is mg.
The results show that the maximum reaction rate (V) of the pullulanase of the present invention on the pullulan substratemax) Catalytic constant (K)cat) Substrate affinity (K)m) And catalytic efficiency (K)cat/Km) Respectively 489.6U/mg-1、844.9s-1、0.19mg·mL-1、4371.0s-1·mg-1mL, which indicates that the pullulanase of the present invention has higher activity.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> Nanjing university of forestry
<120> pullulanase with high secretion capacity and application thereof
<160>10
<170>PatentIn version 3.3
<210>1
<211>937
<212>PRT
<213> Artificial sequence
<400>1
Ala Asp Thr Thr Lys Leu Thr Ile His Tyr Gln Pro Ala Ser Asn Asp
1 5 10 15
Thr Lys Glu Trp Gly Leu Trp Val Phe Pro Glu Gly Gly Glu Gly Lys
20 25 30
Pro Tyr Ala Phe Thr Gly Glu Asp Gln Phe Gly Lys Val Ala Glu Val
35 40 45
Glu Leu Pro Gly Thr Tyr Asp Lys Val Gly Phe Ile Val Arg Thr Glu
50 55 60
Ser Trp Glu Lys Asp Gly Gly Asp Arg Phe Val Ser Val Glu Asn Gly
65 70 75 80
Glu Gly Glu Val Trp Val Lys Ser Gly Asp Glu His Thr Tyr Thr Ser
85 90 95
Pro Pro Asp Gly Glu Tyr Arg Asp Leu Pro Glu Phe Asp Lys Val Asn
100 105 110
Val Thr Leu Asn Tyr His Arg Tyr Asp Gly Asp Tyr Ser Gly Trp Asn
115 120 125
Ile Trp Thr Trp Pro Gly Asp Glu Lys Glu Gly Lys Gln Ile Glu Phe
130 135 140
Thr Glu Asp Thr Asn Phe Gly Lys Lys Ala Thr Tyr Thr Ile Asn Ser
145 150 155 160
Gly Ser Gly Asn Leu Phe Asp Lys Ile Gly Phe Ile Val Arg His Ser
165 170 175
Thr Ser Asp Ser Asp Trp Glu Asn Lys Asp Gly Gly Asn Arg Phe Ile
180 185 190
Thr Lys Val Gly Lys Asp Gly Asn Val Glu Val Trp Ile Val Gln Gly
195 200 205
Gln Asn Arg Ile Tyr Tyr Asp Arg Ala Gly Val Asp Leu Thr Arg Lys
210 215 220
Ile Met Lys Ala Thr Met Asp Thr Phe Asn Gln Ile Thr Leu Glu Thr
225 230 235 240
Asn Val Pro Phe Asp Ser Ser Lys Ser Leu Gly Asp Ile Glu Ile Asp
245 250 255
Gly Ala Gln Ile Glu Lys Val Thr Pro Tyr Lys Glu Gly Gly Asp Ala
260 265 270
Val Thr Thr Lys Ile Lys Ile Ile Thr Lys Arg Pro Leu Asp Val Thr
275 280 285
Gln Thr Tyr Arg Val Lys Gln Gln Gly Tyr Gly Ser Ala Asp Val Val
290 295 300
Asn Gly Asn Val Val Arg Thr Lys Glu Phe Asp Glu Lys Tyr Ala Tyr
305 310 315 320
Ser Gly Asn Asp Leu Gly Asn Thr Tyr Ser Lys Lys Gln Thr Asn Phe
325 330 335
Arg Val Trp Ala Pro Thr Ala Ser Glu Ala Lys Leu Val Thr Tyr Ser
340 345 350
Ser Trp Asn Ser Lys Ala Leu Lys Glu Ile Ser Met Thr Lys Ser Glu
355 360 365
Lys Gly Thr Trp Lys Ala Glu Leu Lys Gly Asn Gln Asp Glu Leu Ile
370 375 380
Tyr Thr Tyr Lys Val Lys Ile Gly Asn Val Trp Asn Glu Ala Val Asp
385 390 395 400
Pro Tyr Val Arg Ala Thr Thr Val Asn Gly Asp Arg Gly Val Val Val
405 410 415
Asp Leu Ala Lys Thr Asn Pro Lys Lys Trp Ser Thr Asn Lys Pro Lys
420 425 430
Phe Lys Asn Pro Glu Asp Ala Ile Ile Tyr Glu Leu His Val Arg Asp
435 440 445
Leu Ser Ser Gln Lys Glu Ser Gly Ile Lys Asn Lys Gly Lys Tyr Leu
450 455 460
Gly Val Ala Glu Trp Asn Thr Lys Gly Pro Asn Gly Val Lys Thr Gly
465 470 475 480
Leu Ser His Ile Lys Asp Leu Gly Val Thr His Val Gln Phe Leu Pro
485 490 495
Ile Tyr Asp Tyr Arg Thr Val Asp Glu Thr Lys Leu Asn Glu Pro Gln
500 505 510
Phe Asn Trp Gly Tyr Asp Pro Lys Asn Tyr Asn Val Pro Glu Gly Ser
515 520 525
Tyr Ser Thr Asn Pro Tyr Asn Pro Lys Thr Arg Ile Ile Glu Leu Lys
530535 540
Gln Met Ile Gln Thr Leu His Asp Lys Gln Leu Arg Met Val Met Asp
545 550 555 560
Val Val Tyr Asn His Val Tyr Ala Val Ser Glu His Ser Phe Asp Lys
565 570 575
Leu Val Pro Gly Tyr Tyr Phe Arg Tyr Lys Glu Asp Gly Thr Leu Ser
580 585 590
Asn Gly Thr Gly Val Gly Asn Asp Thr Ala Ser Glu Arg Lys Met Val
595 600 605
Arg Lys Phe Ile Val Asp Ser Val Ala Tyr Trp Ala Lys Glu Tyr His
610 615 620
Ile Asp Gly Phe Arg Phe Asp Leu Met Gly Ile His Asp Thr Lys Thr
625 630 635 640
Met Asn Glu Val Arg Lys Lys Leu Asp Glu Leu Asp Pro Ser Ile Ile
645 650 655
Val Leu Gly Glu Gly Trp Asp Leu Gly Thr Glu Leu Asp Ala Lys Leu
660 665 670
Lys Ala Asn Gln Lys Asn Ala Gln Asp Met Lys Arg Ile Ala His Phe
675 680 685
Asn Asp Gly Met Arg Asp Gly Leu Lys Gly Ser Val Phe Phe Asp His
690695 700
Asp Asn Gly Phe Val Asn Gly Lys Gln Gly Gln Glu Lys Leu Ile Gln
705 710 715 720
Gln Ser Ile Ala Ala Gly Ile Asp Tyr Asp Arg Ser Thr Ala Thr Tyr
725 730 735
Glu Asp Pro Asp Gln Val Val Thr Tyr Val Glu Ala His Asp Asn His
740 745 750
Thr Leu Trp Asp Lys Leu Gln Leu Thr Asn Pro Ala Asp Thr Glu Gln
755 760 765
Thr Lys Lys Gln Met His Lys Leu Ala Ser Ser Ile Ile Leu Thr Ser
770 775 780
Gln Gly Met Asn Phe Ile His Ala Gly Gln Glu Phe Met Arg Thr Lys
785 790 795 800
Gly Gly Asp His Asn Ser Tyr Gln Ser Pro Asp Ser Val Asn Gln Leu
805 810 815
Asp Trp Lys Arg Arg Ala Ala Phe Arg Gln Glu Val Asn Tyr Met Lys
820 825 830
Gly Leu Ile Ala Leu Arg Lys Gln Tyr Ser Ser Phe Arg Met Thr Ser
835 840 845
Ala Gln Ala Ile His Gln Asn Leu Arg Phe Val Asp Ala Pro Ala Asn
850 855 860
Val Val Gly Tyr Thr Leu Asn Ala Lys Ala Asn Lys Asp Lys Ala Asn
865 870 875 880
Glu Ile Met Val Ile His Asn Ala Asn Lys Gln Ala Gln Thr Val His
885 890 895
Leu Pro Ser Asn Arg Thr Trp Arg Leu Leu Val Asp Gly Glu Arg Ala
900 905 910
Gly Thr Lys Thr Leu Arg Thr Val Lys Gly Asn Thr Ile Asn Val Ser
915 920 925
Pro Leu Ser Thr Phe Val Leu Val Arg
930 935
<210>2
<211>2814
<212>DNA
<213> Artificial sequence
<400>2
gcggatacca caaaactcac gattcactat cagcctgctt caaacgatac aaaagaatgg 60
ggattatggg tttttcctga aggaggagaa gggaagcctt acgcatttac aggggaagat 120
caatttggaa aagtagcgga agttgaacta cccggtactt atgacaaagt aggttttatt 180
gtacgaacag agtcgtggga aaaagatgga ggagaccgct tcgtttcagt ggagaacggt 240
gaaggagaag tatgggtaaa aagtggagat gaacatacat atacgtctcc tcctgacggt 300
gaatatcgag atttaccgga atttgataaa gtaaatgtta ccttaaacta tcatcggtac 360
gatggagatt atagcgggtg gaacatttgg acatggccag gtgatgaaaa agaaggcaag 420
caaatcgaat ttacggaaga cacgaatttt gggaagaaag caacttacac aattaacagt 480
ggaagtggta atttatttga caaaattggt tttattgtac gccattctac tagtgacagc 540
gattgggaaa ataaagacgg aggaaaccgc tttattacca aggttggtaa agatgggaat 600
gtagaagtat ggattgttca agggcaaaat cgaatttatt acgatagagc tggagtagat 660
ctcactcgga aaatcatgaa ggcgacgatg gatacattta atcaaatcac cttagaaaca 720
aacgtgccgt ttgactcttc taagagttta ggagacattg aaattgacgg agctcaaatt 780
gagaaagtga caccttataa agagggagga gacgctgtta ctactaaaat taaaatcata 840
acaaaacggc ctttggacgt tacacaaacg tatagagtta agcaacaagg atacggatca 900
gctgatgtag tcaatggaaa tgtcgtgcgt acgaaagaat ttgacgaaaa gtatgcatat 960
agcggaaacg atttaggaaa cacatattct aagaaacaaa caaattttcg cgtatgggct 1020
ccaactgcaa gcgaagccaa gctagtgact tattcgtcat ggaactcgaa agctttaaaa 1080
gaaatttcta tgactaaaag tgaaaaagga acgtggaaag cggagttaaa aggaaatcaa 1140
gatgaactaa tttatacgta caaagtaaaa attgggaacg tctggaatga agcggttgat 1200
ccatatgtac gagcaacaac ggtcaatgga gatcgcggag tcgtcgtaga tttggctaaa 1260
acaaatccga aaaagtggag tacaaataag ccgaaattta aaaatccaga agatgccatt 1320
atctatgaac tgcacgtacg agatttatcg tctcaaaaag agagcggtat caaaaacaaa 1380
ggaaaatatt taggcgtagc ggagtggaat acgaaaggac caaatggagt aaaaacagga 1440
ctcagtcata ttaaagattt aggtgtaacg cacgtccagt tcctgcctat ttacgattat 1500
cgtacggtgg atgaaacgaa attaaacgag ccgcagttta actggggata tgatccgaaa 1560
aattataacg tcccagaagg ctcctattca acaaacccat ataatcctaa gactagaatt 1620
attgagctca aacaaatgat tcaaacgctt catgacaagc agcttcgcat ggtaatggac 1680
gttgtttata atcacgtgta tgcagtgagt gagcatagtt ttgataaact agttccaggc 1740
tactacttcc gttataaaga agatggaact ctatccaacg gaacaggtgt tggaaacgac 1800
acggcgtctg aacggaaaat ggttcggaag tttatcgtag attctgttgc gtattgggca 1860
aaagagtacc acattgacgg gtttcggttt gatttaatgg ggattcacga tacaaagaca 1920
atgaatgaag tgagaaagaa gttagatgaa cttgatcctt ccattatcgt cttaggagag 1980
ggctgggatc taggaaccga gcttgacgct aagctcaaag ctaaccagaa aaacgctcag 2040
gatatgaagc gcatcgctca ctttaatgac ggtatgcggg atggtcttaa aggcagcgta 2100
ttttttgatc atgacaacgg atttgtgaat ggaaagcaag gacaagaaaa gctcatacag 2160
caaagtatag cagctggaat tgattatgat cgttcaactg ccacgtatga agatccagat 2220
caagttgtta cgtatgtaga agcgcacgat aaccatacgt tatgggataa gcttcagctg 2280
acgaaccctg ctgacaccga gcaaacgaaa aagcaaatgc acaagcttgc ttcttctatt 2340
attttaacat ctcaaggaat gaactttata catgcaggtc aagagtttat gagaacaaaa 2400
ggcggagacc ataacagcta tcagtcaccc gattctgtca atcagctaga ttggaagcgc 2460
cgagcagctt tcagacaaga agtaaattac atgaagggac ttattgcgct tcgaaagcaa 2520
tattcgtcat ttagaatgac gagtgcacag gctattcatc aaaatttacg ctttgttgat 2580
gcaccagcaa acgtagtagg ttatacgtta aatgctaaag ctaataaaga taaagcaaac 2640
gaaataatgg tgattcataa tgcaaataaa caagctcaaa cggttcattt gccttctaac 2700
agaacgtgga gattacttgt tgacggcgag cgggccggaa caaaaacact ccgtacggtg 2760
aaaggaaata caataaacgt ttcgcctctt tcgacatttg tactagtaag ataa 2814
<210>3
<211>36
<212>DNA
<213> Artificial sequence
<400>3
ccggcgatgg ccatggcgga taccacaaaa ctcacg 36
<210>4
<211>37
<212>DNA
<213> Artificial sequence
<400>4
gtgcggccgc aagcttatct tactagtaca aatgtcg 37
<210>5
<211>897
<212>PRT
<213> Artificial sequence
<400>5
Met Trp Pro Tyr Gln Pro Val Asn Gly Asn Gly Ala Ala Tyr Gln Phe
1 5 10 15
Thr Gly Thr Asn Asp Asp Phe Gly Ala Val Ala Asp Thr Gln Val Pro
20 25 30
Gly Asp Asn Thr Gln Val Gly Leu Ile Val Arg Lys Asn Asp Trp Ser
35 40 45
Glu Lys Asn Thr Pro Asn Asp Leu His Ile Asp Leu Ala Lys Gly His
50 55 60
Glu Val Trp Ile Val Gln Gly Asp Pro Thr Ile Tyr Tyr Asn Leu Ser
65 70 75 80
Asp Ala Gln Ala Ala Ala Ile Pro Ser Val Ser Asn Ala Tyr Leu Asp
85 90 95
Asp Glu Lys Thr Val Leu Ala Lys Leu Ser Met Pro Met Thr Leu Ala
100 105 110
Asp Ala Ala Ser Gly Phe Thr Val Ile Asp Lys Thr Thr Gly Glu Lys
115 120 125
Ile Pro Val Thr Ser Ala Val Ser Ala Asn Pro Val Thr Ala Val Leu
130 135 140
Val Gly Asp Leu Gln Gln Ala Leu Gly Ala Ala Asn Asn Trp Ser Pro
145 150 155 160
Asp Asp Asp His Thr Leu Leu Lys Lys Ile Asn Pro Asn Leu Tyr Gln
165 170 175
Leu Ser Gly Thr Leu Pro Ala Gly Thr Tyr Gln Tyr Lys Ile Ala Leu
180 185 190
Asp His Ser Trp Asn Thr Ser Tyr Pro Gly Asn Asn Val Ser Leu Thr
195200 205
Val Pro Gln Gly Gly Glu Lys Val Thr Phe Thr Tyr Ile Pro Ser Thr
210 215 220
Asn Gln Val Phe Asp Ser Val Asn His Pro Asn Gln Ala Phe Pro Thr
225 230 235 240
Ser Ser Ala Gly Val Gln Thr Asn Leu Val Gln Leu Thr Leu Ala Ser
245 250 255
Ala Pro Asp Val Thr His Asn Leu Asp Val Ala Ala Asp Gly Tyr Lys
260 265 270
Ala His Asn Ile Leu Pro Arg Asn Val Leu Asn Leu Pro Arg Tyr Asp
275 280 285
Tyr Ser Gly Asn Asp Leu Gly Asn Val Tyr Ser Lys Asp Ala Thr Ser
290 295 300
Phe Arg Val Trp Ala Pro Thr Ala Ser Asn Val Gln Leu Leu Leu Tyr
305 310 315 320
Asn Ser Glu Lys Gly Ser Ile Thr Lys Gln Leu Glu Met Gln Lys Ser
325 330 335
Asp Asn Gly Thr Trp Lys Leu Gln Val Ser Gly Asn Leu Glu Asn Trp
340 345 350
Tyr Tyr Leu Tyr Gln Val Thr Val Asn Gly Thr Thr Gln Thr Ala Val
355360 365
Asp Pro Tyr Ala Arg Ala Ile Ser Val Asn Ala Thr Arg Gly Met Ile
370 375 380
Val Asp Leu Lys Ala Thr Asp Pro Ala Gly Trp Gln Gly Asp His Glu
385 390 395 400
Gln Thr Pro Ala Asn Pro Val Asp Glu Val Ile Tyr Glu Ala His Val
405 410 415
Arg Asp Phe Ser Ile Asp Ala Asn Ser Gly Met Lys Asn Lys Gly Lys
420 425 430
Tyr Leu Ala Phe Thr Glu His Gly Thr Lys Gly Pro Asp His Val Lys
435 440 445
Thr Gly Ile Asp Ser Leu Lys Glu Leu Gly Ile Thr Thr Val Gln Leu
450 455 460
Gln Pro Val Glu Glu Phe Asn Ser Ile Asp Glu Thr Gln Pro Asp Thr
465 470 475 480
Tyr Asn Trp Gly Tyr Asp Pro Arg Asn Tyr Asn Val Pro Glu Gly Ala
485 490 495
Tyr Ala Thr Thr Pro Glu Gly Thr Ala Arg Ile Thr Glu Leu Lys Gln
500 505 510
Leu Ile Gln Ser Leu His Gln Gln Arg Ile Gly Val Asn Met Asp Val
515520 525
Val Tyr Asn His Thr Phe Asp Val Met Val Ser Asp Phe Asp Lys Ile
530 535 540
Val Pro Gln Tyr Tyr Tyr Arg Thr Asp Ser Asn Gly Asn Tyr Thr Asn
545 550 555 560
Gly Ser Gly Cys Gly Asn Glu Phe Ala Thr Glu His Pro Met Ala Gln
565 570 575
Lys Phe Val Leu Asp Ser Val Asn Tyr Trp Val Asn Glu Tyr His Val
580 585 590
Asp Gly Phe Arg Phe Asp Leu Met Ala Leu Leu Gly Lys Asp Thr Met
595 600 605
Ala Lys Ile Ser Asn Glu Leu His Ala Ile Asn Pro Gly Ile Val Leu
610 615 620
Tyr Gly Glu Pro Trp Thr Gly Gly Thr Ser Gly Leu Ser Ser Asp Gln
625 630 635 640
Leu Val Thr Lys Gly Gln Gln Lys Gly Leu Gly Ile Gly Val Phe Asn
645 650 655
Asp Asn Ile Arg Asn Gly Leu Asp Gly Asn Val Phe Asp Lys Thr Ala
660 665 670
Gln Gly Phe Ala Thr Gly Asp Pro Asn Gln Val Asp Val Ile Lys Asn
675 680685
Gly Val Ile Gly Ser Ile Gln Asp Phe Thr Ser Ala Pro Ser Glu Thr
690 695 700
Ile Asn Tyr Val Thr Ser His Asp Asn Met Thr Leu Trp Asp Lys Ile
705 710 715 720
Leu Ala Ser Asn Pro Ser Asp Thr Glu Ala Asp Arg Ile Lys Met Asp
725 730 735
Glu Leu Ala His Ala Val Val Phe Thr Ser Gln Gly Val Pro Phe Met
740 745 750
Gln Gly Gly Glu Glu Met Leu Arg Thr Lys Gly Gly Asn Asp Asn Ser
755 760 765
Tyr Asn Ala Gly Asp Ser Val Asn Gln Phe Asp Trp Ser Arg Lys Ala
770 775 780
Gln Phe Lys Asp Val Phe Asp Tyr Phe Ser Ser Met Ile His Leu Arg
785 790 795 800
Asn Gln His Pro Ala Phe Arg Met Thr Thr Ala Asp Gln Ile Lys Gln
805 810 815
Asn Leu Thr Phe Leu Glu Ser Pro Thr Asn Thr Val Ala Phe Glu Leu
820 825 830
Lys Asn Tyr Ala Asn His Asp Thr Trp Lys Asn Ile Ile Val Met Tyr
835 840845
Asn Pro Asn Lys Thr Ser Gln Thr Leu Asn Leu Pro Ser Gly Asp Trp
850 855 860
Thr Ile Val Gly Leu Gly Asp Gln Ile Gly Glu Lys Ser Leu Gly His
865 870 875 880
Val Met Gly Asn Val Gln Val Pro Ala Ile Ser Thr Leu Ile Leu Lys
885 890 895
Gln
<210>6
<211>2766
<212>DNA
<213> Artificial sequence
<400>6
gacagcacca gtaccaaggt catcgtccac taccacagat tcgacagtaa ttacaccaac 60
tgggatgttt ggatgtggcc ataccaacca gtcaacggaa acggtgctgc atatcagttt 120
actggaacca acgacgactt cggtgcagtc gctgacactc aggttccagg tgacaacacc 180
caggtcggtc tcatcgtcag aaagaacgac tggtctgaga agaacactcc taatgacttg 240
cacatcgact tggctaaggg tcacgaggtc tggatcgtcc agggtgatcc taccatctac 300
tataaccttt ctgatgctca agcagctgcc atccctagcg tcagcaacgc ttacttggac 360
gacgagaaga ctgtcttggc taaactgtcc atgccaatga ctcttgctga cgcagcctct 420
ggtttcactg tcatcgacaa gactactgga gagaagattc cagttactag cgcagtcagt 480
gccaacccag tcactgctgt cttggtcggt gatcttcagc aagcacttgg tgctgctaac 540
aactggagtc ctgacgacga ccataccttgttgaagaaaa tcaaccctaa cttgtatcag 600
cttagtggca ccttgcctgc tggcacttat cagtacaaaa tcgctcttga tcacagctgg 660
aacaccagtt accctggcaa taacgtctcc ttgaccgtcc cacaaggtgg tgagaaagtc 720
actttcactt acatccctag cactaaccaa gtctttgact ctgttaacca cccaaatcag 780
gcttttccaa ccagtagtgc tggtgttcaa accaaccttg ttcagcttac ccttgcttct 840
gctcctgacg ttactcacaa cctggacgtc gctgctgatg gctataaggc tcataacatc 900
cttccaagaa acgtcctgaa ccttccaaga tacgactact ctggcaacga ccttggcaac 960
gtctacagca aagacgctac cagtttcaga gtctgggcac ctactgccag taacgttcaa 1020
ctcttgctct ataactccga gaagggaagt atcaccaagc aacttgagat gcagaagtcc 1080
gacaatggaa cctggaagct ccaagtcagt ggcaacttgg agaattggta ctacttgtac 1140
caggttactg tcaatggtac tacccagact gctgttgacc cttacgctag agccatcagt 1200
gtcaacgcta ccagaggcat gatcgttgat ctcaaggcta ctgatccagc tggttggcaa 1260
ggagaccacg agcaaactcc agctaaccct gttgacgagg tcatctacga agctcacgtc 1320
agagacttca gtatcgacgc aaacagtggc atgaagaaca aaggtaagta ccttgccttc 1380
actgaacatg gcaccaaagg tcctgatcac gtcaaaactg gcatcgactc tctcaaagag 1440
cttggtatta ctaccgtcca gcttcagcca gtcgaagagt tcaactccat cgacgaaact 1500
caaccagaca cttacaattg gggttatgac cctagaaact acaatgttcc tgaaggtgcc 1560
tacgctacca ctcctgaggg tactgctaga atcaccgaac tcaaacagct catccagtcc 1620
ttgcaccaac aaagaatcgg tgtcaacatg gacgtcgtct acaaccacactttcgacgtc 1680
atggtcagcg acttcgacaa gatcgttcca cagtactact acagaactga ctccaacggt 1740
aactacacta atggctctgg ctgtggtaac gagtttgcta ctgaacatcc tatggctcag 1800
aagttcgtct tggactccgt caactactgg gtcaacgaat atcatgttga cggtttcaga 1860
ttcgatctta tggccttgct tggcaaggat actatggcta agatcagcaa cgaacttcac 1920
gctatcaatc caggcatcgt cctttacggt gagccttgga ctggtggcac cagtggtctt 1980
agctctgatc agttggtcac taaaggccag cagaaaggtc ttggcatcgg tgtctttaac 2040
gacaacatca gaaacggttt ggacggtaat gtcttcgaca agactgctca gggtttcgct 2100
actggtgatc ctaaccaagt tgacgtcatc aagaacggtg tcattggctc catccaggac 2160
ttcaccagtg ctccatctga gaccatcaat tacgtcactt ctcacgacaa tatgactttg 2220
tgggacaaga tccttgcttc caacccttcc gataccgaag cagatagaat caagatggac 2280
gagcttgctc acgctgtcgt ctttaccagt cagggtgttc ctttcatgca gggaggtgag 2340
gagatgctta gaaccaaagg tggtaacgac aattcctaca atgctggtga ctccgtcaac 2400
cagtttgatt ggagtagaaa ggctcagttc aaggacgtct tcgactattt cagctccatg 2460
atccacttga gaaaccaaca tccagctttc agaatgacca ctgctgacca gatcaagcag 2520
aacttgacct ttcttgagtc tcctaccaat actgtcgcat ttgaactcaa gaactacgct 2580
aatcacgaca cctggaagaa catcatcgtt atgtacaatc ctaacaaaac cagtcagact 2640
ttgaacttgc cttctggtga ctggactatc gtcggtcttg gtgatcagat cggagaaaag 2700
agccttggac acgtcatggg taacgtccag gtcccagcca tctccacttt gatccttaag 2760
cagtaa 2766
<210>7
<211>937
<212>PRT
<213> Artificial sequence
<400>7
Ala Asp Ser Thr Lys Ile Thr Ile His Tyr Gln Pro Ala Ser Asn Asp
1 5 10 15
Thr Lys Glu Trp Gly Leu Trp Val Phe Pro Glu Gly Gly Glu Gly Lys
20 25 30
Ala Tyr Ala Phe Thr Gly Glu Asp Gln Phe Gly Lys Val Ala Glu Val
35 40 45
Glu Leu Pro Gly Thr Tyr Asp Lys Val Gly Phe Ile Val Arg Thr Glu
50 55 60
Ser Trp Glu Lys Asp Gly Gly Asp Arg Phe Val Ser Val Glu Asn Gly
65 70 75 80
Glu Gly Glu Val Trp Val Lys Ser Gly Asp Glu His Thr Tyr Thr Ser
85 90 95
Pro Pro Asp Gly Glu Tyr Arg Asp Leu Pro Lys Phe Asp Lys Val Asn
100 105 110
Val Thr Leu Asn Tyr His Arg Tyr Asp Gly Asp Tyr Ser Gly Trp Asn
115 120 125
Ile Trp Thr Trp Pro Gly AspGlu Lys Glu Gly Lys Gln Ile Gln Phe
130 135 140
Thr Glu Asp Thr Asn Phe Gly Lys Arg Ala Thr Tyr Thr Leu Asn Ser
145 150 155 160
Gly Thr Gly Asn Leu Phe Asp Lys Ile Gly Phe Ile Val Arg His Ser
165 170 175
Thr Ser Asp Ser Asp Trp Glu Asn Lys Asp Gly Gly Asp Arg Phe Ile
180 185 190
Thr Lys Val Gly Lys Asp Gly Asn Val Glu Val Trp Ile Val Gln Gly
195 200 205
Gln Asn Arg Ile Tyr Tyr Asp Arg Ala Val Val Asp Ile Thr Arg Lys
210 215 220
Ile Thr Lys Ala Thr Met Asp Ala Phe Asn Gln Ile Thr Leu Glu Thr
225 230 235 240
Asn Val Pro Phe Asp Ser Ser Lys Ser Val Gly Asp Ile Glu Ile Asp
245 250 255
Gly Ala Gln Ile Glu Lys Val Thr Pro Tyr Lys Glu Gly Gly Ala Ser
260 265 270
Val Thr Thr Lys Val Lys Ile Ile Thr Lys Gln Pro Leu Asp Val Thr
275 280 285
Lys Thr Tyr Lys Val Lys Gln Lys GlyTyr Gly Ser Ala Asn Val Val
290 295 300
Asn Gly Asn Val Val Arg Thr Lys Glu Phe Asp Glu Lys Tyr Ala Tyr
305 310 315 320
Ser Gly Asn Asp Leu Gly Asn Thr Tyr Ser Lys Lys Gln Thr Asn Phe
325 330 335
Arg Val Trp Ala Pro Thr Ala Ser Glu Ala Lys Leu Val Thr Tyr Ser
340 345 350
Ser Trp Asn Ser Lys Ala Val Lys Glu Ile Ser Met Thr Lys Ser Glu
355 360 365
Lys Gly Thr Trp Lys Ala Glu Leu Lys Gly Asn Gln Asp Glu Leu Ile
370 375 380
Tyr Thr Tyr Lys Val Lys Ile Gly Asn Ser Trp Asn Glu Ala Val Asp
385 390 395 400
Pro Tyr Val Arg Ala Thr Thr Val Asn Gly Asp Arg Gly Val Val Val
405 410 415
Asp Leu Ala Lys Thr Asn Pro Arg Lys Trp Ser Thr Asn Lys Pro Lys
420 425 430
Phe Lys Asn Pro Glu Asp Ala Ile Ile Tyr Glu Leu His Val Arg Asp
435 440 445
Leu Ser Ser Gln Lys Glu Ser Gly Ile Lys AsnLys Gly Lys Tyr Leu
450 455 460
Gly Val Ala Glu Trp Asn Thr Lys Gly Pro Asn Gly Val Lys Thr Gly
465 470 475 480
Leu Ser His Ile Lys Asp Leu Gly Val Thr His Val Gln Phe Leu Pro
485 490 495
Ile Tyr Asp Tyr Arg Thr Val Asp Glu Thr Lys Leu Asn Glu Pro Gln
500 505 510
Phe Asn Trp Gly Tyr Asp Pro Lys Asn Tyr Asn Val Pro Glu Gly Ser
515 520 525
Tyr Ser Thr Asn Pro Tyr Asn Pro Lys Thr Arg Ile Ile Glu Leu Lys
530 535 540
Gln Met Ile Gln Thr Leu His Asp Lys Gln Leu Arg Met Val Met Asp
545 550 555 560
Val Val Tyr Asn His Val Tyr Ala Val Ser Glu His Ser Phe Asp Lys
565 570 575
Leu Val Pro Gly Tyr Tyr Phe Arg Tyr Lys Glu Asp Gly Thr Leu Ser
580 585 590
Asn Gly Thr Gly Val Gly Asn Asp Thr Ala Ser Glu Arg Lys Met Val
595 600 605
Arg Lys Phe Ile Val Asp Ser Val Ala Tyr Trp Ala LysGlu Tyr His
610 615 620
Ile Asp Gly Phe Arg Phe Asp Leu Met Gly Ile His Asp Thr Lys Thr
625 630 635 640
Met Asn Glu Val Arg Lys Lys Leu Asp Glu Met Asp Pro Ser Ile Ile
645 650 655
Ile Leu Gly Glu Gly Trp Asp Leu Gly Thr Glu Leu Asp Ala Lys Leu
660 665 670
Lys Ala Asn Gln Lys Asn Ala Gln Asp Met Lys Arg Ile Ala His Phe
675 680 685
Asn Asp Gly Met Arg Asp Gly Leu Lys Gly Ser Val Phe Phe Asp His
690 695 700
Asp Asn Gly Phe Val Asn Gly Lys Gln Gly Gln Glu Lys Leu Ile Gln
705 710 715 720
Gln Ser Ile Ala Ala Gly Ile Asp Tyr Asp Arg Ser Thr Ala Thr Tyr
725 730 735
Glu Asp Pro Asp Gln Val Val Thr Tyr Val Glu Ala His Asp Asn His
740 745 750
Thr Leu Trp Asp Lys Leu Gln Leu Thr Asn Pro Ala Asp Thr Glu Arg
755 760 765
Thr Arg Met Gln Met His Lys Leu Ala Ser Ser Ile Thr Leu ThrSer
770 775 780
Gln Gly Met Asn Phe Ile His Ala Gly Gln Glu Phe Met Arg Thr Lys
785 790 795 800
Gly Gly Asp His Asn Ser Tyr Gln Ser Pro Asp Ser Val Asn Gln Leu
805 810 815
Asp Trp Lys Arg Arg Ala Ala Phe Ser Gln Glu Val Asp Tyr Met Lys
820 825 830
Gly Leu Ile Ala Leu Arg Lys Gln Tyr Ser Ser Phe Arg Met Thr Ser
835 840 845
Ala Gln Ala Ile His Gln Asn Leu Arg Phe Val Asp Ala Pro Ala Asn
850 855 860
Val Val Gly Tyr Thr Leu Asn Ala Lys Ala Asn Lys Asp Lys Ala Asn
865 870 875 880
Glu Ile Met Val Ile His Asn Ala Asn Lys Gln Ala Gln Thr Val His
885 890 895
Leu Pro Ser Asn Arg Thr Trp Arg Leu Leu Val Asp Gly Glu Arg Ala
900 905 910
Gly Thr Gln Thr Leu Arg Thr Val Lys Gly Asn Thr Val Asn Val Ser
915 920 925
Pro Leu Ser Thr Phe Val Leu Val Arg
930 935
<210>8
<211>2814
<212>DNA
<213> Artificial sequence
<400>8
gcagactcaa caaaaatcac gattcactat cagcctgctt caaacgacac aaaagaatgg 60
ggattatggg tttttcctga aggtggagaa gggaaggctt acgcatttac aggggaagat 120
cagtttggaa aagtagcgga agttgaacta cccggtactt atgataaagt aggctttatt 180
gtacgaacag agtcatggga aaaagatgga ggagaccgct tcgtctcagt ggagaacggt 240
gaaggagaag tatgggtaaa aagtggagat gaacatacat atacgtctcc tcctgacggt 300
gaataccggg atttaccgaa gtttgataaa gtaaacgtta ctttaaacta tcaccgatat 360
gacggagatt acagcgggtg gaacatttgg acgtggccag gtgacgaaaa agaaggcaag 420
cagatccaat ttacggaaga cacgaatttt gggaagagag ccacttacac gcttaacagt 480
ggaactggta acttatttga caaaataggt tttattgtac gccactctac tagtgatagc 540
gattgggaaa ataaagacgg aggagaccgc tttattacca aggttgggaa agatgggaat 600
gtagaagtat ggattgttca agggcaaaat cgaatttatt atgatagagc tgtagtagat 660
atcactcgga aaatcacgaa agcgactatg gatgcattta atcaaattac attagaaaca 720
aacgtaccgt ttgactcttc taagagtgta ggagacattg aaattgacgg agctcaaatt 780
gagaaagtga caccttataa agagggagga gcctctgtta ctactaaagt taaaatcata 840
acaaaacagc ctctggacgt tacaaaaacg tataaagtta agcaaaaagg atacggctca 900
gctaatgtag tcaatggaaa tgtcgttcgt accaaagaat ttgacgaaaa gtatgcgtac 960
agcgggaatg atttaggaaa cacgtattct aagaaacaaa caaattttcg cgtatgggct 1020
ccaaccgcaa gcgaagccaa gctagtgact tattcttcat ggaactccaa agctgtaaaa 1080
gaaatttcta tgactaaaag tgaaaaagga acgtggaaag cggagttaaa aggaaatcaa 1140
gatgagctaa tttacacgta caaagtaaaa attgggaaca gctggaatga agcggttgat 1200
ccatatgtac gagcaacaac ggtgaatgga gaccgtggag ttgtggtgga tttggctaag 1260
acaaatccaa gaaagtggag tacgaataag ccaaaattta aaaatccaga agatgccatc 1320
atctatgaac tacacgtaag agatttatcg tctcaaaaag aaagcggtat caaaaataaa 1380
ggaaagtatt tgggtgtagc ggagtggaat acgaagggac caaatggcgt aaaaacagga 1440
ctcagtcata ttaaagacct aggtgtaacg catgttcagt ttctgcctat ttatgattat 1500
cgtacggtgg atgaaacgaa attaaacgag ccgcagttta actggggata tgatccgaaa 1560
aattataacg taccagaagg ttcctattca acaaatccat ataatcctaa gactagaatt 1620
attgagctta aacaaatgat tcaaacgctt catgacaagc agcttcgtat ggtaatggac 1680
gttgtctata accatgtgta tgcagtgagt gagcatagct ttgataaact agttccaggc 1740
tactacttcc gctataaaga agatggaact ctatccaacg gaacaggtgt tggaaacgac 1800
acggcttctg aacggaaaat ggttcggaag tttattgtag attctgtggc ttattgggca 1860
aaagagtacc acattgacgg gtttcgattt gatttaatgg ggattcacga tacaaagaca 1920
atgaatgaag tgagaaagaa gttagatgaa atggatcctt ccattatcat cttaggagag 1980
ggatgggatc taggaaccga gcttgacgct aagctcaaag ctaaccagaa aaacgctcag 2040
gatatgaaac gcatcgctca ctttaatgac ggtatgcgag atggccttaa aggcagcgtg 2100
ttttttgatc atgacaacgg atttgttaat ggaaagcaag gacaagaaaa gctcatacag 2160
caaagtatag cagctggaat agattacgat cgttcaactg ccacgtatga agatccagat 2220
caagttgtca catatgtaga agcgcacgac aaccatacgt tatgggataa gcttcagctg 2280
acgaaccctg ctgacacgga gcgaacgaga atgcagatgc ataagcttgc ttcttccatt 2340
actttaacat ctcaaggaat gaactttata catgcaggtc aagagtttat gagaacaaaa 2400
ggcggagatc ataatagcta tcagtcaccc gattctgtca atcagttaga ttggaagcgc 2460
cgagcagctt tcagccaaga agtagattac atgaagggac ttattgcgct tcgaaagcaa 2520
tattcctcat ttagaatgac aagtgcacag gctattcatc aaaatttacg ctttgttgat 2580
gcaccagcaa acgtagtagg ttatacgtta aatgctaaag ccaataaaga taaagcaaac 2640
gaaataatgg tgattcataa tgcaaataaa caagctcaaa cagttcattt accttctaac 2700
agaacgtgga gattacttgt tgacggcgag cgggcaggaa cacaaacgct ccgtacggtg 2760
aaaggaaata cagtaaacgt ttcgcctctt tcgacatttg tgctagtaag ataa 2814
<210>9
<211>926
<212>PRT
<213> Artificial sequence
<400>9
Asp Gly Asn Thr Thr Asn Ile Val Val His Tyr Phe Arg Pro Ser Gly
1 5 10 15
Asp Tyr Thr Asp Trp Asn Leu Trp Met Trp Pro Glu Asn Gly Asp Gly
20 25 30
Ala Glu Tyr Asp Phe Asn Gln Pro Thr Asp Ser Tyr Gly Glu Val Ala
35 40 45
Ser Val Asp Ile Pro Gly Asn Pro Ser Gln Val Gly Ile Ile Val Arg
50 55 60
Lys Gly Asn Trp Asp Ala Lys Asp Ile Asp Ser Asp Arg Tyr Ile Asp
65 70 75 80
Leu Ser Lys Gly His Glu Ile Trp Leu Val Gln Gly Asn Ser Gln Ile
85 90 95
Phe Tyr Ser Glu Lys Asp Ala Glu Ala Ala Ala Gln Pro Ala Val Ser
100 105 110
Asn Ala Tyr Leu Asp Ala Ser Asn Gln Val Leu Val Lys Leu Ser Gln
115 120 125
Pro Phe Thr Leu Gly Glu Gly Ser Ser Gly Phe Thr Val His Asp Asp
130 135 140
Thr Ala Asn Lys Asp Ile Pro Val Thr Ser Val Ser Asp Ala Asn Gln
145 150 155 160
Val Thr Ala Val Leu Ala Gly Thr Phe Gln His Ile Phe Gly Gly Ser
165 170 175
Asp Trp Ala Pro Asp Asn His Asn Thr Leu Leu Lys Lys Val Asn Ser
180 185 190
Asn Leu Tyr Gln Phe Ser Gly Asn Leu Pro Glu Gly Asn Tyr Gln Tyr
195 200 205
Lys Val Ala Leu Asn Asp Ser Trp Asn Asn Pro Ser Tyr Pro Ser Asp
210 215 220
Asn Ile Asn Leu Thr Val Pro Ala Gly Gly Ala His Val Thr Phe Ser
225 230 235 240
Tyr Ile Pro Ser Thr His Ala Val Tyr Asp Thr Ile Asn Asn Pro Asn
245 250 255
Ala Asp Leu Gln Val Asp Ser Ser Gly Val Lys Thr Asp Leu Val Ala
260 265 270
Val Thr Leu Gly Glu Asn Pro Asp Val Ser His Thr Leu Ser Ile Gln
275 280 285
Thr Glu Asp Tyr Gln Ala Gly Gln Val Ile Pro Arg Lys Val Leu Asp
290 295 300
Ser Ser Gln Tyr Tyr Tyr Ser Gly Asp Asp Leu Gly Asn Thr Tyr Thr
305 310 315 320
Lys Asn Ala Thr Thr Phe Lys Val Trp Ala Pro Thr Ser Thr Gln Val
325 330 335
Asn Val Leu Leu Tyr Asn Ser Ala Thr Gly Ala Val Thr Lys Thr Val
340 345 350
Pro Met Thr Ala Ser Gly His Gly Val Trp Glu Ala Thr Val Asn Gln
355 360 365
Asp Leu Glu Asn Trp Tyr Tyr Met Tyr Glu Val Thr Gly Gln Gly Ser
370 375 380
Thr Arg Thr Ala Val Asp Pro Tyr Ala Thr Ala Ile Ala Pro Asn Gly
385 390 395 400
Thr Arg Gly Met Ile Val Asp Leu Ala Lys Thr Asp Pro Ala Gly Trp
405 410 415
Glu Ser Asp Lys His Ile Thr Pro Lys Asn Ile Glu Asp Glu Val Ile
420 425 430
Tyr Glu Met Asp Val Arg Asp Phe Ser Ile Asp Ser Asn Ser Gly Met
435 440 445
Lys Asn Lys Gly Lys Tyr Leu Ala Leu Thr Glu Lys Gly Thr Lys Gly
450 455 460
Pro Asp Asn Val Lys Thr Gly Val Asp Ser Leu Lys Gln Leu Gly Ile
465 470 475 480
Thr His Val Gln Leu Gln Pro Val Phe Ala Phe Asn Ser Val Asn Glu
485 490 495
Asn Asp Pro Thr Gln Tyr Asn Trp Gly Tyr Asp Pro Arg Asn Tyr Asn
500 505 510
Val Pro Glu Gly Gln Tyr Ala Thr Asn Ala Asn Gly Thr Thr Arg Ile
515 520 525
Lys Glu Phe Lys Glu Met Val Leu Ser Leu His Gln Asp His Ile Gly
530 535 540
Val Asn Met Asp Val Val Tyr Asn His Thr Phe Ala Thr Gln Ile Ser
545 550 555 560
Asp Phe Asp Lys Ile Val Pro Glu Tyr Tyr Tyr Arg Thr Asp Asp Ala
565 570 575
Gly Asn Tyr Thr Asn Gly Ser Gly Thr Gly Asn Glu Ile Ala Ala Glu
580 585 590
Arg Pro Met Val Gln Lys Phe Ile Ile Asp Ser Leu Lys Phe Trp Val
595 600 605
Asn Glu Tyr His Val Asp Gly Phe Arg Phe Asp Leu Met Ala Leu Leu
610 615 620
Gly Lys Asp Thr Met Ser Lys Ala Ala Thr Gln Leu His Ala Ile Asp
625 630 635 640
Pro Gly Ile Ala Leu Tyr Gly Glu Pro Trp Thr Gly Gly Thr Ser Ala
645 650 655
Leu Pro Ala Asp Gln Leu Leu Thr Lys Gly Ala Gln Lys Gly Met Gly
660 665 670
Val Ala Val Phe Asn Asp Asn Leu Arg Asn Gly Leu Asp Gly Ser Val
675 680 685
Phe Asp Ser Ser Ala Gln Gly Phe Ala Thr Gly Ala Thr Gly Leu Thr
690 695 700
Asp Ala Ile Lys Asn Gly Val Glu Gly Ser Ile Asn Asp Phe Thr Ala
705 710 715 720
Ser Pro Gly Glu Thr Ile Asn Tyr Val Thr Ser His Asp Asn Tyr Thr
725 730 735
Leu Trp Asp Lys Ile Ala Gln Ser Asn Pro Asn Asp Ser Glu Ala Asp
740 745 750
Arg Ile Lys Met Asp Glu Leu Ala Gln Ala Ile Val Met Thr Ser Gln
755 760 765
Gly Ile Pro Phe Met Gln Gly Gly Glu Glu Met Leu Arg Thr Lys Gly
770 775 780
Gly Asn Asp Asn Ser Tyr Asn Ala Gly Asp Val Val Asn Glu Phe Asp
785 790 795 800
Trp Ser Arg Lys Ala Gln Tyr Pro Asp Val Phe Asn Tyr Tyr Ser Gly
805 810 815
Leu Ile His Leu Arg Leu Asp His Pro Ala Phe Arg Met Thr Thr Ala
820 825 830
Asn Glu Ile Asn Ser His Leu Gln Phe Leu Asn Ser Pro Glu Asn Thr
835 840 845
Val Ala Tyr Glu Leu Ser Asp His Ala Asn Lys Asp Thr Trp Gly Asn
850 855 860
Ile Val Val Ile Tyr Asn Pro Asn Lys Thr Ala Glu Thr Ile Asn Leu
865 870 875 880
Pro Ser Gly Lys Trp Glu Ile Asn Ala Thr Ser Gly Lys Val Gly Glu
885 890 895
Ser Thr Leu Gly Gln Ala Glu Gly Ser Val Gln Val Pro Gly Ile Ser
900 905 910
Met Met Ile Leu His Gln Glu Val Ser Pro Ser Asp Gly Lys
915 920 925
<210>10
<211>2781
<212>DNA
<213> Artificial sequence
<400>10
gatgggaaca ccacaaacat cgtagtccat tattttcgtc ctagtgggga ttatacggat 60
tggaatcttt ggatgtggcc ggagaacggt gatggggctg agtatgattt taatcaaccg 120
actgattctt atggggaggt tgcaagtgtg gacattcctg gaaacccaag tcaagtaggg 180
attattgtcc gtaaaggaaa ttgggatgcg aaagacattg atagtgaccg ctacatcgat 240
ttaagcaaag ggcatgagat ttggctcgtc caaggaaaca gccagatttt ctatagtgaa 300
aaggatgctg aggcagccgc acaacctgct gtaagtaacg cttatttaga tgcttccaac 360
caagtgttgg tcaagcttag ccagccgttt actcttggtg aaggttcaag cggttttacg 420
gttcatgatg acacagcaaa taaggatatt ccagttacat ctgttagtga tgccaatcag 480
gtaacggctg ttttagcagg tactttccag catatttttg gggggagtga ttgggcaccg 540
gataatcaca atactttact aaaaaaggtg aatagcaatc tctatcaatt ttcaggaaat 600
cttcctgaag gaaactacca atataaagtg gctttaaatg atagctggaa taatccgagc 660
tacccatctg ataacattaa tttgacagtg ccagctggtg gtgcccatgt tacattttct 720
tatataccat ccacccatgc tgtttatgac acgattaaca atcctaatgc ggatttacaa 780
gtagatagca gcggtgttaa gacggatctc gtggcggtta ctcttggaga aaatcctgat 840
gtaagccata ccctgtccat tcaaacagag gactatcagg caggacaggt catacctcgt 900
aaggtgcttg attcatccca gtactactat tccggagatg atctcgggaa tacctataca 960
aagaatgcaa ctacctttaa ggtctgggcg cctacatcca ctcaagtaaa tgtccttctt 1020
tataatagtg caaccggcgc ggtaactaaa acggttccaa tgaccgcatc aggccatggt 1080
gtatgggaag caacagtcaa ccaagacctt gaaaattggt attacatgta tgaggtaaca 1140
ggacaaggct caacccgaac ggctgttgat ccgtatgcaa cagctattgc accaaacgga 1200
acgagaggca tgattgtgga cctagccaaa acagacccgg ccggatggga gagtgacaaa 1260
catattacgc caaagaatat agaagatgaa gtcatctatgaaatggatgt tcgtgacttt 1320
tccatcgact ctaattcggg tatgaaaaat aaaggaaagt atttggcact tacagaaaaa 1380
ggaactaaag gccctgacaa tgtaaagaca ggggtagatt ccttaaaaca acttgggatt 1440
actcatgttc agctgcagcc tgttttcgca tttaatagtg tcaatgaaaa cgatccaact 1500
caatataatt ggggttatga ccctcgcaac tacaatgttc ctgagggaca atatgctact 1560
aatgcaaacg gaacaactcg gattaaagag tttaaggaaa tggttctttc actccatcag 1620
gaccacattg gggttaatat ggatgttgtt tataatcata cctttgccac gcaaatctct 1680
gacttcgata agattgtgcc agaatattac taccgcacgg atgatgctgg taactacact 1740
aacggctcag gtactggaaa cgaaatcgca gccgaaagac caatggttca aaaatttatt 1800
atcgattcac ttaagttttg ggtcaatgag taccacgttg acggtttccg ttttgactta 1860
atggcgttgc ttggaaaaga tacaatgtct aaagctgcca cgcagcttca tgccattgat 1920
ccaggaattg ctctctacgg tgagccatgg acaggaggaa catccgcgct gccagccgat 1980
cagcttttaa caaaaggagc tcaaaaaggc atgggagtgg ctgtatttaa tgacaatctg 2040
cgaaacggtt tggacggcag tgtctttgat tcatctgctc aaggttttgc gacaggtgct 2100
actggtttaa cggatgctat taaaaatgga gttgaaggaa gtattaatga cttcaccgct 2160
tcaccaggcg agacgatcaa ctatgtcaca agtcatgata actataccct ttgggacaag 2220
attgcccaaa gcaatccaaa cgattctgaa gcggatcgaa ttaaaatgga tgagctcgct 2280
caagcgatcg tcatgacctc acaaggcatt cctttcatgc agggcgggga agaaatgctt 2340
cgtacgaaag gcggcaacga caatagctat aatgctggtg atgtagtgaa cgagtttgat 2400
tggagcagaa aagctcaata tccagatgtt ttcaattatt atagcgggct gattcatctt 2460
cgtcttgatc acccagcctt ccgcatgacg acagctaatg aaatcaatag ccacctccaa 2520
ttcctaaata gcccagagaa cacagtggcc tatgaattat ctgatcatgc aaataaagat 2580
acatggggta atattgtggt tatttataat ccaaataaaa cggcagaaac cattaatttg 2640
ccaagcggga aatgggaaat caatgcgacg agcggtaagg tgggagaatc cacacttggt 2700
caagcagagg gcagtgttca agttccaggc atatctatga tgattcttca tcaagaagta 2760
agcccatctg atggtaaata g 2781