CN109112166A - Enzyme process prepares ticagrelor midbody - Google Patents

Enzyme process prepares ticagrelor midbody Download PDF

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CN109112166A
CN109112166A CN201710496576.5A CN201710496576A CN109112166A CN 109112166 A CN109112166 A CN 109112166A CN 201710496576 A CN201710496576 A CN 201710496576A CN 109112166 A CN109112166 A CN 109112166A
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val
leu
ser
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CN109112166B (en
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马宏敏
田振华
程占冰
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Yikelai Biotechnology (Group) Co.,Ltd.
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Shanghai Yi Ke Lai Biological Medicine Science And Technology Co Ltd
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The present invention provides a kind of methods of enzyme process preparation ticagrelor midbody, specifically, the method includes the steps: it uses KRED enzyme as catalyst, is reacted with compound 28 with reducing agent, obtain compound 29.Wherein, the KRED enzyme has the protein sequence as shown in SEQ ID NO:3-SEQ ID NO:16.The method can obtain compound 29, and products therefrom can be used for the preparation of subsequent products without further purification in high yield with high selectivity.

Description

Enzyme process prepares ticagrelor midbody
Technical field
The present invention relates to medical compounds fields, and specifically, the present invention provides a kind of enzyme process to prepare among Ticagrelor The method of body.
Background technique
Ticagrelor is the anticoagulation medicine of AstraZeneca exploitation, and U.S. FDA approval listing in 2011 has good city Field prospect.United States Patent (USP) US7250419 reports its synthetic route, and as follows, intermediate 10 is three passes in API synthesis One of key intermediate, economic value with higher:
The conventional synthetic route of intermediate 10 is as follows, and menthol is used for Asymmetric cyclopropanation as chiral auxiliary, Cost is very high (US7067663).
Patent EP2589587A1 reports the route by the preparation of nitroketone 23 10, and KRED is as catalyst, but product 24 yield only has 63%, and ee value only has 80%.
Another route of EP2589587A1 report is as follows, and feature is using CBS catalyst, and BH3 restores to obtain chiral chlorine Alcohol 29, but ee value, in 90-93%, ee value is not high, product needs to refine to improve ee value, and price is higher.
In conclusion there is an urgent need in the art to low cost, mild condition, the method for selective highland prepare compound 10.
Summary of the invention
The object of the present invention is to provide a kind of low cost, mild condition, the methods of selective highland prepare compound 10.
The first aspect of the present invention provides a kind of method of enzyme process preparation ticagrelor midbody, the method includes Step:
It uses KRED enzyme as catalyst, is reacted with compound 28 with reducing agent, obtain compound 29:
Wherein, the KRED enzyme is the alcohol dehydrogenase from Leifsonia aquatica ATCC 14665, and its Mutant；Or the KRED enzyme has the protein sequence as shown in SEQ ID NO:15 or SEQ ID NO:16.
In another preferred example, the alcohol dehydrogenase from Leifsonia aquatica ATCC 14665 Mutant is carried out in site selected from the group below: T100R, T100K, S148I, S148V, S148L, or combinations thereof.
In another preferred example, the alcohol dehydrogenase from Leifsonia aquatica ATCC 14665 has Just like protein sequence shown in SEQ ID NO:3-SEQ ID NO:14.
In another preferred example, the initial concentration of the compound 28 is 10g/L-200g/L.
In another preferred example, the KRED enzyme dosage is 5-50g/L.
In another preferred example, in the reaction, the reducing agent is selected from the group: isopropanol, glucose or its group It closes.
In another preferred example, the reaction carries out in the presence of dehydrogenase, preferably exists in glucose dehydrogenase Lower progress.
In another preferred example, the reaction carries out in the presence of co-factor；Preferably, the co-factor is selected from down Group: reducibility coenzyme NAD+, NADP+, or combinations thereof.
In another preferred example, the dosage of the reducibility coenzyme NAD+ is 0-10mM, preferably 0.1-10mM.
In another preferred example, the reaction carries out in the transformation system of pH value 6.5~7.5.
In another preferred example, the expression vector is selected from the group: plasmid, bacteriophage, adenovirus vector, reverse transcription disease Poisonous carrier.
A kind of expression vector, which is characterized in that the expression vector includes the DNA sequence as shown in SEQ ID NO:31 Column.
The fourth aspect of the present invention provides a kind of hydro-reduction method, which comprises uses KRED enzyme as urging Agent；Wherein, the KRED enzyme is the alcohol dehydrogenase from Leifsonia aquatica ATCC 14665, and its prominent Variant；Or the KRED enzyme has the protein sequence as shown in SEQ ID NO:15 or SEQ ID NO:16；
Preferably, the method includes the steps that as follows:
It using compound I as raw material, is catalyzed with KRED enzyme, obtains Formula II compound；Wherein, R1It is selected from the group: replacing Or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 naphthenic base, substituted or unsubstituted C6-C10 aryl；
R2Be selected from the group: halogen, substituted or unsubstituted C3-C6 naphthenic base, takes substituted or unsubstituted C1-C6 alkyl Generation or unsubstituted C6-C10 aryl；
N=1,2,3,4 or 5；
The substitution refers to that one or more hydrogen atoms on group are replaced by substituent group selected from the group below: halogen, C1~ C4 alkyl, C1~C4 halogenated alkyl, hydroxyl, amino, nitro, carboxyl, C1~C4 alkoxy, C1~C4 halogenated alkoxy, cyano.
In another preferred example, the alcohol dehydrogenase from Leifsonia aquatica ATCC 14665 Mutant is mutated in following site: T100R, T100K, S148I, S148V, S148L, or combinations thereof.
In another preferred example, the alcohol dehydrogenase from Leifsonia aquatica ATCC 14665 has Just like protein sequence shown in SEQ ID NO:3-SEQ ID NO:14.
The fifth aspect of the present invention provides a kind of preparation method of compound 10, the method includes the steps: (1) it uses First aspect present invention or fourth aspect it is any as described in method be prepared compound 29, and (2) with the compound 29 are prepared compound 10:
The sixth aspect of the present invention provides a kind of preparation method of compound Ticagrelor, which is characterized in that the side Method comprising steps of
(1) with first aspect present invention or fourth aspect it is any as described in method compound 29 is prepared, and
(2) Ticagrelor is prepared with the compound 29.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.
Specific embodiment
The present inventor's in-depth study by long-term provides a kind of KRED enzyme for being used to prepare compound 29.It is described Enzyme can highly selective (ee value > 99%) 29 compound of formula is prepared, reduce costs, reduce refining step, have There is very big economic value.Based on above-mentioned discovery, inventor completes the present invention.
Term
As used herein, " halogen " refers to F, Cl, Br and I.More preferably, halogen atom is selected from F, Cl and Br.
As used herein, " C1-C6 alkyl " refers to the alkyl of the linear chain or branched chain including 1-6 carbon atom, such as methyl, Ethyl, propyl, isopropyl, butyl, isobutyl group, tert-butyl or similar group.
Term " C3-C6 naphthenic base " refers to the naphthenic base comprising 3-6 carbon atom, such as cyclopropyl, cyclobutyl, ring penta Base, cyclohexyl or similar group.
Term " C6-C10 aryl " refers to the aryl with 6-10 carbon atom, including monocycle or aryl bicyclic, such as phenyl, Naphthalene, or similar group.
The KRED enzyme of prepare compound 29
Ticagrelor midbody 10 is one of three key intermediates in API synthesis, in existing preparation route, hand Property chloropharin 29 be the committed step for introducing chiral centre, therefore, high efficiency and the intermediate is obtained with high selectivity, for preparation Compound 10 or even compound Ticagrelor are all of great significance.However, existing preparation method is difficult to expeditiously obtain The intermediate.Therefore, the present invention determines to develop the side that enzyme catalysis method prepares chiral chloropharin 29 on the basis of prior art route Method.
There is the enzyme catalyst of reduction activation by screening, obtained a series of reductase with catalysis activity, such as Enzyme shown in SEQ ID NO:3-16.Wherein, the SEQ ID NO:3-14 is from Leifsonia aquatica The alcohol dehydrogenase and its mutant of ATCC 14665；Or the KRED enzyme has such as SEQ ID NO:15 or SEQ ID NO:16 Shown in protein sequence.Above-mentioned KRED enzyme can achieve up to 95% or more ee value, therefore product is without being further purified It can be used as the starting material of next step.
On the basis of above-mentioned KRED enzyme, the preparation system of chiral chloropharin 29 is obtained.In the transformant of pH value 6.5~7.5 It is substrate with chlorine ketone 28 in system, ketoreductase KRED is catalyst, and NAD+ is co-factor, and isopropanol or glucose are hydrogen donor, It carries out asymmetric reduction reaction and generates chiral chloropharin 29, ee > 99% of part KRED enzyme.
The initial substrate concentration is 10g/L-200g/L, and KRED dosage is 5-50g/L, the use of reducibility coenzyme NAD+ Amount is 0-10mM.
Product Compound 29 obtained is used directly for reacting in next step, such as is prepared by step as follows Compound 10:
Obtained compound 10 can be also used for further preparing Ticagrelor.
Ketone group hydro-reduction enzyme
Since KRED enzyme of the invention has substrate universality, it can be used for a series of ketone group hydrogenation of substrates also It is former.On this basis, the present invention also provides a kind of hydro-reduction methods, which comprises uses KRED enzyme as catalyst； Wherein, the KRED enzyme is the alcohol dehydrogenase and its mutant from Leifsonia aquatica ATCC 14665； Or the KRED enzyme has the protein sequence as shown in SEQ ID NO:15 or SEQ ID NO:16.Preferably, the side Method includes the steps that as follows:
It using compound I as raw material, is catalyzed with KRED enzyme, obtains Formula II compound；Wherein, it is described wherein, it is described KRED enzyme be from Leifsonia aquatica ATCC 14665 alcohol dehydrogenase and its mutant；Or it is described KRED enzyme has the protein sequence as shown in SEQ ID NO:15 or SEQ ID NO:16.
Wherein, R1Be selected from the group: substituted or unsubstituted C1-C6 alkyl, takes substituted or unsubstituted C3-C6 naphthenic base Generation or unsubstituted C6-C10 aryl；
R2Be selected from the group: halogen, substituted or unsubstituted C3-C6 naphthenic base, takes substituted or unsubstituted C1-C6 alkyl Generation or unsubstituted C6-C10 aryl；
N=1,2,3,4 or 5.
The substitution refers to that one or more hydrogen atoms on group are replaced by substituent group selected from the group below: halogen, C1~ C4 alkyl, C1~C4 halogenated alkyl, hydroxyl, amino, nitro, carboxyl, C1~C4 alkoxy, C1~C4 halogenated alkoxy, cyano.
In another preferred example, R2For halogen, and n=2.
It preferably further include reducing agent in the reaction, a kind of preferred reducing agent is selected from the group: isopropanol, grape Sugar, or combinations thereof.
In another preferred example, the reaction carries out in the presence of dehydrogenase, preferably exists in glucose dehydrogenase Lower progress.
In another preferred example, the reaction carries out in the presence of co-factor；Preferably, the co-factor is selected from down Group: reducibility coenzyme NAD+, NADP+, or combinations thereof.
In another preferred example, the initial concentration of the substrate compound of formula I is 10g/L-200g/L.
In another preferred example, the KRED enzyme dosage is 5-50g/L.
In another preferred example, the dosage of the reducibility coenzyme NAD+ is 0-10mM.
Compared with prior art, main advantages of the present invention include:
It (1), can be with during above-mentioned enzyme is reacted for aromatic ring α carbonyl reduction the present invention provides a kind of KRED serial enzymes Reach very high yield and selectivity (ee value can be with > 99%).
(2) the present invention provides a kind of method of safe and efficient prepare compound 29, above-mentioned method is matched without chirality Body and expensive dangerous borine, friendly process, and the dosage of organic solvent reduces, product yield and selectivity are high, products therefrom Without further refining a series of intermediate that can be prepared used as compounds.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are calculated by weight.
The building of 1 strain of embodiment and high density fermentation
The DNA sequence dna (sequence such as following table) of composite coding protein sequence 3-16, and be connected to pET28a NdeI and The site HindIII obtains the escherichia coli plasmid KRED03-16 containing KRED3-16.Plasmid is converted into e. coli bl21 (DE3), corresponding strain is obtained on the Kana resistance LB plate containing 30ug/mL, picking individual colonies are inoculated into LB culture medium In, 37 degree are incubated overnight, and save strain with 20% glycerol.
Strain is inoculated in the 1L shaking flask equipped with 200mL LB culture medium, in 37 DEG C, 180-220rpm cultivates 10- 16h.Above-mentioned cultured seed is inoculated on 3L (glucose 4g/ in tank fermentation medium (M9) in the ratio of 10% (v/v) L, disodium hydrogen phosphate 12.8g/L, potassium dihydrogen phosphate 3g/L, ammonium chloride 1g/L, sodium sulphate 0.5g/L, calcium chloride 0.0152g/L, six Aqueous magnesium chloride 0.41g/L), it is cultivated under conditions of 25-35 DEG C, 300-800 rpm, air mass flow 2-6L/min.Cultivate 6-10h Afterwards, the supplemented medium containing 60% glycerol is added with the rate stream of 5-20mL/h, continued to fermentation ends.Flow feeding culture radix Hour to OD600When reaching 80-100, tank is put, thalline were collected by centrifugation by 5000rpm.
The KRED of the screening anti-prelog rule of embodiment 2~6
Using homemade KRED serial enzymes as catalyst, using following reaction system, the concentration of chlorine ketone 28 is 0.1M, NAD+ Concentration be 0.02mM, glucose 0.15M, KRED freeze-dried powder 10g/L, glucose dehydrogenase freeze-dried powder 5g/L.Reaction temperature: 30 DEG C, the reaction time: 4h, between 1M NaOH control pH value 6.5~7.5.After reaction in three times with 2 times of volumes of acetic acid Ethyl ester extraction, dry revolving obtain 29 crude product of S- chloropharin.HPLC detects reaction conversion ratio, and chiral HPLC detects ee value.
Table 1KRED zymoprotein and DNA sequence dna correspond to table
The applicant has been unexpectedly discovered that, compared with similar KRED reductase, the KRED03-16 of the application can be obtained Product configuration in contrast, and good product selectivity are obtained, can be directly used in and prepare in next step without further purification.
The amplification preparation S- chloropharin 29 of embodiment 7
In 1L reaction system, the concentration of chlorination ketone 100g, NAD+ are 0.1g, glucose dehydrogenase freeze-dried powder 5g, the application KRED1410g, glucose 120g.Reaction temperature: 30 DEG C, the reaction time: 8h, conversion ratio 90%；16h, conversion ratio 99%.With 1M NaOH controls pH value 6.5~7.5.It being extracted in three times with 2 times of volumes toluenes after reaction, dry back spin steams solvent, Obtain S- chloropharin 95g, yield 94%, ee value 99%.
Embodiment 8 makees reducing agent with isopropanol
In 1L reaction system, the concentration of chlorination ketone 100g, NAD+ are 0.1g, KRED14-10g, isopropanol 150mL.Reaction Temperature: 30 DEG C, the reaction time: 8h, conversion ratio 82%；16h, conversion ratio 95%.Control pH value 6.5~7.5.Divide after reaction It is extracted three times with 2 times of volumes toluenes, dry back spin steams solvent, obtains S- chloropharin 92g, yield 91%, ee value 99%.
The KRED14 reductone 23 of embodiment 9
Patent EP2589587A1 reports the route by the preparation of nitroketone 23 10, and KRED is as catalyst, but product 24 yield only has 63%, and ee value only has 80%.We make catalyst with enzyme KRED14, and 23 are used as substrate, stereoselectivity Reduction obtains 24.Condition is as follows: the concentration of ketone 23 is 0.1M, and the concentration of NAD+ is 0.02mM, glucose 0.15M, KRED freeze-drying Powder 10g/L, glucose dehydrogenase freeze-dried powder 5g/L.Reaction temperature: 30 DEG C, the reaction time: 16h controls pH value with 1M NaOH Between 6.5~7.5.After reaction with being extracted in three times with 2 times of volume of ethylacetate, it is thick that dry revolving obtains S- chloropharin 29 Product.HPLC detects reaction conversion ratio > 98%, and chiral HPLC detects ee value, ee value > 98%.
Embodiment 10:
HPLC condition uses the monitoring reaction of high performance liquid chromatography (HPLC) method: with water and acetonitrile (45:55) for mobile phase, color Spectrum column is ODS-18 reversed-phase column, and Shimadzu LC-15C high performance liquid chromatography detects UV absorption under 210nm；Reaction system water and Acetonitrile (45:55) is diluted, sample detection after being centrifuged and being filtered with nylon membrane.In the present invention preferably reaction system, HPLC detects reaction process: after reaction 1 hour, detection, 10.3min is reduzate alcohol, and 17.5min is raw ketone.
Chiral purity is analyzed using 1260 liquid chromatogram of Agilent, testing conditions are as follows: Chiralpak AD-H column, N-hexane: ethyl alcohol=90:10,0.8mL/min, Detection wavelength 220nm.Retention time S- configuration: 8.1min；R- configuration: 11.6min。
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.
Sequence table
<110>Shanghai Yi Kelai biological medicine Science and Technology Ltd.
<120>enzyme process prepares ticagrelor midbody
<130> P2016-2308
<160> 32
<170> PatentIn version 3.5
<210> 1
<211> 252
<212> PRT
<213>artificial sequence
<400> 1
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Tyr Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Val
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Thr
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ser Cys Val Leu
85 90 95
Lys Ser Val Lys Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Glu Gly Leu Val Gly Asp Pro Ser Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys
180 185 190
Thr Gln Arg Leu Asp Gln Leu Glu Gly Trp Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Val Thr Pro Met Gly Arg Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 2
<211> 252
<212> PRT
<213>artificial sequence
<400> 2
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg Arg Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Glu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp His Glu Gly Leu Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 3
<211> 251
<212> PRT
<213>artificial sequence
<400> 3
Met Ala Gln Tyr Asp Val Ala Asp Arg Ser Ala Ile Val Thr Gly Gly
1 5 10 15
Gly Ser Gly Ile Gly Arg Ala Val Ala Leu Thr Leu Ala Ala Ser Gly
20 25 30
Ala Ala Val Leu Val Thr Asp Leu Asn Glu Glu His Ala Gln Ala Val
35 40 45
Val Ala Glu Ile Glu Ala Ala Gly Gly Lys Ala Ala Ala Leu Ala Gly
50 55 60
Asp Val Thr Asp Pro Ala Phe Gly Glu Ala Ser Val Ala Ala Ala Asn
65 70 75 80
Ala Leu Ala Pro Leu Lys Ile Ala Val Asn Asn Ala Gly Ile Gly Gly
85 90 95
Glu Ala Ala Thr Val Gly Asp Tyr Ser Leu Asp Ser Trp Arg Lys Val
100 105 110
Ile Glu Val Asn Leu Asn Ala Val Phe Tyr Gly Met Gln Pro Gln Leu
115 120 125
Lys Ala Met Ala Ala Asn Gly Gly Gly Ala Ile Val Asn Met Ala Ser
130 135 140
Ile Leu Gly Ser Val Gly Phe Ala Asn Ser Ser Ala Tyr Val Thr Ala
145 150 155 160
Lys His Ala Leu Leu Gly Leu Thr Gln Asn Ala Ala Leu Glu Tyr Ala
165 170 175
Ala Asp Lys Val Arg Val Val Ala Val Gly Pro Gly Phe Ile Arg Thr
180 185 190
Pro Leu Val Glu Ala Asn Leu Ser Ala Glu Ala Leu Ala Phe Leu Glu
195 200 205
Gly Lys His Ala Leu Gly Arg Leu Gly Glu Pro Glu Glu Val Ala Ser
210 215 220
Leu Val Ala Phe Leu Ala Ser Asp Ala Ala Ser Phe Ile Thr Gly Ser
225 230 235 240
Tyr His Leu Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 4
<211> 251
<212> PRT
<213>artificial sequence
<400> 4
Met Ala Gln Tyr Asp Val Ala Asp Arg Ser Ala Ile Val Thr Gly Gly
1 5 10 15
Gly Ser Gly Ile Gly Arg Ala Val Ala Leu Thr Leu Ala Ala Ser Gly
20 25 30
Ala Ala Val Leu Val Thr Asp Leu Asn Glu Glu His Ala Gln Ala Val
35 40 45
Val Ala Glu Ile Glu Ala Ala Gly Gly Lys Ala Ala Ala Leu Ala Gly
50 55 60
Asp Val Thr Asp Pro Ala Phe Gly Glu Ala Ser Val Ala Ala Ala Asn
65 70 75 80
Ala Leu Ala Pro Leu Lys Ile Ala Val Asn Asn Ala Gly Ile Gly Gly
85 90 95
Glu Ala Ala Arg Val Gly Asp Tyr Ser Leu Asp Ser Trp Arg Lys Val
100 105 110
Ile Glu Val Asn Leu Asn Ala Val Phe Tyr Gly Met Gln Pro Gln Leu
115 120 125
Lys Ala Met Ala Ala Asn Gly Gly Gly Ala Ile Val Asn Met Ala Ser
130 135 140
Ile Leu Gly Ser Val Gly Phe Ala Asn Ser Ser Ala Tyr Val Thr Ala
145 150 155 160
Lys His Ala Leu Leu Gly Leu Thr Gln Asn Ala Ala Leu Glu Tyr Ala
165 170 175
Ala Asp Lys Val Arg Val Val Ala Val Gly Pro Gly Phe Ile Arg Thr
180 185 190
Pro Leu Val Glu Ala Asn Leu Ser Ala Glu Ala Leu Ala Phe Leu Glu
195 200 205
Gly Lys His Ala Leu Gly Arg Leu Gly Glu Pro Glu Glu Val Ala Ser
210 215 220
Leu Val Ala Phe Leu Ala Ser Asp Ala Ala Ser Phe Ile Thr Gly Ser
225 230 235 240
Tyr His Leu Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 5
<211> 251
<212> PRT
<213>artificial sequence
<400> 5
Met Ala Gln Tyr Asp Val Ala Asp Arg Ser Ala Ile Val Thr Gly Gly
1 5 10 15
Gly Ser Gly Ile Gly Arg Ala Val Ala Leu Thr Leu Ala Ala Ser Gly
20 25 30
Ala Ala Val Leu Val Thr Asp Leu Asn Glu Glu His Ala Gln Ala Val
35 40 45
Val Ala Glu Ile Glu Ala Ala Gly Gly Lys Ala Ala Ala Leu Ala Gly
50 55 60
Asp Val Thr Asp Pro Ala Phe Gly Glu Ala Ser Val Ala Ala Ala Asn
65 70 75 80
Ala Leu Ala Pro Leu Lys Ile Ala Val Asn Asn Ala Gly Ile Gly Gly
85 90 95
Glu Ala Ala Lys Val Gly Asp Tyr Ser Leu Asp Ser Trp Arg Lys Val
100 105 110
Ile Glu Val Asn Leu Asn Ala Val Phe Tyr Gly Met Gln Pro Gln Leu
115 120 125
Lys Ala Met Ala Ala Asn Gly Gly Gly Ala Ile Val Asn Met Ala Ser
130 135 140
Ile Leu Gly Ser Val Gly Phe Ala Asn Ser Ser Ala Tyr Val Thr Ala
145 150 155 160
Lys His Ala Leu Leu Gly Leu Thr Gln Asn Ala Ala Leu Glu Tyr Ala
165 170 175
Ala Asp Lys Val Arg Val Val Ala Val Gly Pro Gly Phe Ile Arg Thr
180 185 190
Pro Leu Val Glu Ala Asn Leu Ser Ala Glu Ala Leu Ala Phe Leu Glu
195 200 205
Gly Lys His Ala Leu Gly Arg Leu Gly Glu Pro Glu Glu Val Ala Ser
210 215 220
Leu Val Ala Phe Leu Ala Ser Asp Ala Ala Ser Phe Ile Thr Gly Ser
225 230 235 240
Tyr His Leu Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 6
<211> 251
<212> PRT
<213>artificial sequence
<400> 6
Met Ala Gln Tyr Asp Val Ala Asp Arg Ser Ala Ile Val Thr Gly Gly
1 5 10 15
Gly Ser Gly Ile Gly Arg Ala Val Ala Leu Thr Leu Ala Ala Ser Gly
20 25 30
Ala Ala Val Leu Val Thr Asp Leu Asn Glu Glu His Ala Gln Ala Val
35 40 45
Val Ala Glu Ile Glu Ala Ala Gly Gly Lys Ala Ala Ala Leu Ala Gly
50 55 60
Asp Val Thr Asp Pro Ala Phe Gly Glu Ala Ser Val Ala Ala Ala Asn
65 70 75 80
Ala Leu Ala Pro Leu Lys Ile Ala Val Asn Asn Ala Gly Ile Gly Gly
85 90 95
Glu Ala Ala Thr Val Gly Asp Tyr Ser Leu Asp Ser Trp Arg Lys Val
100 105 110
Ile Glu Val Asn Leu Asn Ala Val Phe Tyr Gly Met Gln Pro Gln Leu
115 120 125
Lys Ala Met Ala Ala Asn Gly Gly Gly Ala Ile Val Asn Met Ala Ser
130 135 140
Ile Leu Gly Ile Val Gly Phe Ala Asn Ser Ser Ala Tyr Val Thr Ala
145 150 155 160
Lys His Ala Leu Leu Gly Leu Thr Gln Asn Ala Ala Leu Glu Tyr Ala
165 170 175
Ala Asp Lys Val Arg Val Val Ala Val Gly Pro Gly Phe Ile Arg Thr
180 185 190
Pro Leu Val Glu Ala Asn Leu Ser Ala Glu Ala Leu Ala Phe Leu Glu
195 200 205
Gly Lys His Ala Leu Gly Arg Leu Gly Glu Pro Glu Glu Val Ala Ser
210 215 220
Leu Val Ala Phe Leu Ala Ser Asp Ala Ala Ser Phe Ile Thr Gly Ser
225 230 235 240
Tyr His Leu Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 7
<211> 251
<212> PRT
<213>artificial sequence
<400> 7
Met Ala Gln Tyr Asp Val Ala Asp Arg Ser Ala Ile Val Thr Gly Gly
1 5 10 15
Gly Ser Gly Ile Gly Arg Ala Val Ala Leu Thr Leu Ala Ala Ser Gly
20 25 30
Ala Ala Val Leu Val Thr Asp Leu Asn Glu Glu His Ala Gln Ala Val
35 40 45
Val Ala Glu Ile Glu Ala Ala Gly Gly Lys Ala Ala Ala Leu Ala Gly
50 55 60
Asp Val Thr Asp Pro Ala Phe Gly Glu Ala Ser Val Ala Ala Ala Asn
65 70 75 80
Ala Leu Ala Pro Leu Lys Ile Ala Val Asn Asn Ala Gly Ile Gly Gly
85 90 95
Glu Ala Ala Thr Val Gly Asp Tyr Ser Leu Asp Ser Trp Arg Lys Val
100 105 110
Ile Glu Val Asn Leu Asn Ala Val Phe Tyr Gly Met Gln Pro Gln Leu
115 120 125
Lys Ala Met Ala Ala Asn Gly Gly Gly Ala Ile Val Asn Met Ala Ser
130 135 140
Ile Leu Gly Val Val Gly Phe Ala Asn Ser Ser Ala Tyr Val Thr Ala
145 150 155 160
Lys His Ala Leu Leu Gly Leu Thr Gln Asn Ala Ala Leu Glu Tyr Ala
165 170 175
Ala Asp Lys Val Arg Val Val Ala Val Gly Pro Gly Phe Ile Arg Thr
180 185 190
Pro Leu Val Glu Ala Asn Leu Ser Ala Glu Ala Leu Ala Phe Leu Glu
195 200 205
Gly Lys His Ala Leu Gly Arg Leu Gly Glu Pro Glu Glu Val Ala Ser
210 215 220
Leu Val Ala Phe Leu Ala Ser Asp Ala Ala Ser Phe Ile Thr Gly Ser
225 230 235 240
Tyr His Leu Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 8
<211> 251
<212> PRT
<213>artificial sequence
<400> 8
Met Ala Gln Tyr Asp Val Ala Asp Arg Ser Ala Ile Val Thr Gly Gly
1 5 10 15
Gly Ser Gly Ile Gly Arg Ala Val Ala Leu Thr Leu Ala Ala Ser Gly
20 25 30
Ala Ala Val Leu Val Thr Asp Leu Asn Glu Glu His Ala Gln Ala Val
35 40 45
Val Ala Glu Ile Glu Ala Ala Gly Gly Lys Ala Ala Ala Leu Ala Gly
50 55 60
Asp Val Thr Asp Pro Ala Phe Gly Glu Ala Ser Val Ala Ala Ala Asn
65 70 75 80
Ala Leu Ala Pro Leu Lys Ile Ala Val Asn Asn Ala Gly Ile Gly Gly
85 90 95
Glu Ala Ala Thr Val Gly Asp Tyr Ser Leu Asp Ser Trp Arg Lys Val
100 105 110
Ile Glu Val Asn Leu Asn Ala Val Phe Tyr Gly Met Gln Pro Gln Leu
115 120 125
Lys Ala Met Ala Ala Asn Gly Gly Gly Ala Ile Val Asn Met Ala Ser
130 135 140
Ile Leu Gly Leu Val Gly Phe Ala Asn Ser Ser Ala Tyr Val Thr Ala
145 150 155 160
Lys His Ala Leu Leu Gly Leu Thr Gln Asn Ala Ala Leu Glu Tyr Ala
165 170 175
Ala Asp Lys Val Arg Val Val Ala Val Gly Pro Gly Phe Ile Arg Thr
180 185 190
Pro Leu Val Glu Ala Asn Leu Ser Ala Glu Ala Leu Ala Phe Leu Glu
195 200 205
Gly Lys His Ala Leu Gly Arg Leu Gly Glu Pro Glu Glu Val Ala Ser
210 215 220
Leu Val Ala Phe Leu Ala Ser Asp Ala Ala Ser Phe Ile Thr Gly Ser
225 230 235 240
Tyr His Leu Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 9
<211> 251
<212> PRT
<213>artificial sequence
<400> 9
Met Ala Gln Tyr Asp Val Ala Asp Arg Ser Ala Ile Val Thr Gly Gly
1 5 10 15
Gly Ser Gly Ile Gly Arg Ala Val Ala Leu Thr Leu Ala Ala Ser Gly
20 25 30
Ala Ala Val Leu Val Thr Asp Leu Asn Glu Glu His Ala Gln Ala Val
35 40 45
Val Ala Glu Ile Glu Ala Ala Gly Gly Lys Ala Ala Ala Leu Ala Gly
50 55 60
Asp Val Thr Asp Pro Ala Phe Gly Glu Ala Ser Val Ala Ala Ala Asn
65 70 75 80
Ala Leu Ala Pro Leu Lys Ile Ala Val Asn Asn Ala Gly Ile Gly Gly
85 90 95
Glu Ala Ala Arg Val Gly Asp Tyr Ser Leu Asp Ser Trp Arg Lys Val
100 105 110
Ile Glu Val Asn Leu Asn Ala Val Phe Tyr Gly Met Gln Pro Gln Leu
115 120 125
Lys Ala Met Ala Ala Asn Gly Gly Gly Ala Ile Val Asn Met Ala Ser
130 135 140
Ile Leu Gly Ile Val Gly Phe Ala Asn Ser Ser Ala Tyr Val Thr Ala
145 150 155 160
Lys His Ala Leu Leu Gly Leu Thr Gln Asn Ala Ala Leu Glu Tyr Ala
165 170 175
Ala Asp Lys Val Arg Val Val Ala Val Gly Pro Gly Phe Ile Arg Thr
180 185 190
Pro Leu Val Glu Ala Asn Leu Ser Ala Glu Ala Leu Ala Phe Leu Glu
195 200 205
Gly Lys His Ala Leu Gly Arg Leu Gly Glu Pro Glu Glu Val Ala Ser
210 215 220
Leu Val Ala Phe Leu Ala Ser Asp Ala Ala Ser Phe Ile Thr Gly Ser
225 230 235 240
Tyr His Leu Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 10
<211> 251
<212> PRT
<213>artificial sequence
<400> 10
Met Ala Gln Tyr Asp Val Ala Asp Arg Ser Ala Ile Val Thr Gly Gly
1 5 10 15
Gly Ser Gly Ile Gly Arg Ala Val Ala Leu Thr Leu Ala Ala Ser Gly
20 25 30
Ala Ala Val Leu Val Thr Asp Leu Asn Glu Glu His Ala Gln Ala Val
35 40 45
Val Ala Glu Ile Glu Ala Ala Gly Gly Lys Ala Ala Ala Leu Ala Gly
50 55 60
Asp Val Thr Asp Pro Ala Phe Gly Glu Ala Ser Val Ala Ala Ala Asn
65 70 75 80
Ala Leu Ala Pro Leu Lys Ile Ala Val Asn Asn Ala Gly Ile Gly Gly
85 90 95
Glu Ala Ala Arg Val Gly Asp Tyr Ser Leu Asp Ser Trp Arg Lys Val
100 105 110
Ile Glu Val Asn Leu Asn Ala Val Phe Tyr Gly Met Gln Pro Gln Leu
115 120 125
Lys Ala Met Ala Ala Asn Gly Gly Gly Ala Ile Val Asn Met Ala Ser
130 135 140
Ile Leu Gly Val Val Gly Phe Ala Asn Ser Ser Ala Tyr Val Thr Ala
145 150 155 160
Lys His Ala Leu Leu Gly Leu Thr Gln Asn Ala Ala Leu Glu Tyr Ala
165 170 175
Ala Asp Lys Val Arg Val Val Ala Val Gly Pro Gly Phe Ile Arg Thr
180 185 190
Pro Leu Val Glu Ala Asn Leu Ser Ala Glu Ala Leu Ala Phe Leu Glu
195 200 205
Gly Lys His Ala Leu Gly Arg Leu Gly Glu Pro Glu Glu Val Ala Ser
210 215 220
Leu Val Ala Phe Leu Ala Ser Asp Ala Ala Ser Phe Ile Thr Gly Ser
225 230 235 240
Tyr His Leu Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 11
<211> 251
<212> PRT
<213>artificial sequence
<400> 11
Met Ala Gln Tyr Asp Val Ala Asp Arg Ser Ala Ile Val Thr Gly Gly
1 5 10 15
Gly Ser Gly Ile Gly Arg Ala Val Ala Leu Thr Leu Ala Ala Ser Gly
20 25 30
Ala Ala Val Leu Val Thr Asp Leu Asn Glu Glu His Ala Gln Ala Val
35 40 45
Val Ala Glu Ile Glu Ala Ala Gly Gly Lys Ala Ala Ala Leu Ala Gly
50 55 60
Asp Val Thr Asp Pro Ala Phe Gly Glu Ala Ser Val Ala Ala Ala Asn
65 70 75 80
Ala Leu Ala Pro Leu Lys Ile Ala Val Asn Asn Ala Gly Ile Gly Gly
85 90 95
Glu Ala Ala Arg Val Gly Asp Tyr Ser Leu Asp Ser Trp Arg Lys Val
100 105 110
Ile Glu Val Asn Leu Asn Ala Val Phe Tyr Gly Met Gln Pro Gln Leu
115 120 125
Lys Ala Met Ala Ala Asn Gly Gly Gly Ala Ile Val Asn Met Ala Ser
130 135 140
Ile Leu Gly Leu Val Gly Phe Ala Asn Ser Ser Ala Tyr Val Thr Ala
145 150 155 160
Lys His Ala Leu Leu Gly Leu Thr Gln Asn Ala Ala Leu Glu Tyr Ala
165 170 175
Ala Asp Lys Val Arg Val Val Ala Val Gly Pro Gly Phe Ile Arg Thr
180 185 190
Pro Leu Val Glu Ala Asn Leu Ser Ala Glu Ala Leu Ala Phe Leu Glu
195 200 205
Gly Lys His Ala Leu Gly Arg Leu Gly Glu Pro Glu Glu Val Ala Ser
210 215 220
Leu Val Ala Phe Leu Ala Ser Asp Ala Ala Ser Phe Ile Thr Gly Ser
225 230 235 240
Tyr His Leu Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 12
<211> 251
<212> PRT
<213>artificial sequence
<400> 12
Met Ala Gln Tyr Asp Val Ala Asp Arg Ser Ala Ile Val Thr Gly Gly
1 5 10 15
Gly Ser Gly Ile Gly Arg Ala Val Ala Leu Thr Leu Ala Ala Ser Gly
20 25 30
Ala Ala Val Leu Val Thr Asp Leu Asn Glu Glu His Ala Gln Ala Val
35 40 45
Val Ala Glu Ile Glu Ala Ala Gly Gly Lys Ala Ala Ala Leu Ala Gly
50 55 60
Asp Val Thr Asp Pro Ala Phe Gly Glu Ala Ser Val Ala Ala Ala Asn
65 70 75 80
Ala Leu Ala Pro Leu Lys Ile Ala Val Asn Asn Ala Gly Ile Gly Gly
85 90 95
Glu Ala Ala Lys Val Gly Asp Tyr Ser Leu Asp Ser Trp Arg Lys Val
100 105 110
Ile Glu Val Asn Leu Asn Ala Val Phe Tyr Gly Met Gln Pro Gln Leu
115 120 125
Lys Ala Met Ala Ala Asn Gly Gly Gly Ala Ile Val Asn Met Ala Ser
130 135 140
Ile Leu Gly Ile Val Gly Phe Ala Asn Ser Ser Ala Tyr Val Thr Ala
145 150 155 160
Lys His Ala Leu Leu Gly Leu Thr Gln Asn Ala Ala Leu Glu Tyr Ala
165 170 175
Ala Asp Lys Val Arg Val Val Ala Val Gly Pro Gly Phe Ile Arg Thr
180 185 190
Pro Leu Val Glu Ala Asn Leu Ser Ala Glu Ala Leu Ala Phe Leu Glu
195 200 205
Gly Lys His Ala Leu Gly Arg Leu Gly Glu Pro Glu Glu Val Ala Ser
210 215 220
Leu Val Ala Phe Leu Ala Ser Asp Ala Ala Ser Phe Ile Thr Gly Ser
225 230 235 240
Tyr His Leu Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 13
<211> 251
<212> PRT
<213>artificial sequence
<400> 13
Met Ala Gln Tyr Asp Val Ala Asp Arg Ser Ala Ile Val Thr Gly Gly
1 5 10 15
Gly Ser Gly Ile Gly Arg Ala Val Ala Leu Thr Leu Ala Ala Ser Gly
20 25 30
Ala Ala Val Leu Val Thr Asp Leu Asn Glu Glu His Ala Gln Ala Val
35 40 45
Val Ala Glu Ile Glu Ala Ala Gly Gly Lys Ala Ala Ala Leu Ala Gly
50 55 60
Asp Val Thr Asp Pro Ala Phe Gly Glu Ala Ser Val Ala Ala Ala Asn
65 70 75 80
Ala Leu Ala Pro Leu Lys Ile Ala Val Asn Asn Ala Gly Ile Gly Gly
85 90 95
Glu Ala Ala Lys Val Gly Asp Tyr Ser Leu Asp Ser Trp Arg Lys Val
100 105 110
Ile Glu Val Asn Leu Asn Ala Val Phe Tyr Gly Met Gln Pro Gln Leu
115 120 125
Lys Ala Met Ala Ala Asn Gly Gly Gly Ala Ile Val Asn Met Ala Ser
130 135 140
Ile Leu Gly Val Val Gly Phe Ala Asn Ser Ser Ala Tyr Val Thr Ala
145 150 155 160
Lys His Ala Leu Leu Gly Leu Thr Gln Asn Ala Ala Leu Glu Tyr Ala
165 170 175
Ala Asp Lys Val Arg Val Val Ala Val Gly Pro Gly Phe Ile Arg Thr
180 185 190
Pro Leu Val Glu Ala Asn Leu Ser Ala Glu Ala Leu Ala Phe Leu Glu
195 200 205
Gly Lys His Ala Leu Gly Arg Leu Gly Glu Pro Glu Glu Val Ala Ser
210 215 220
Leu Val Ala Phe Leu Ala Ser Asp Ala Ala Ser Phe Ile Thr Gly Ser
225 230 235 240
Tyr His Leu Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 14
<211> 251
<212> PRT
<213>artificial sequence
<400> 14
Met Ala Gln Tyr Asp Val Ala Asp Arg Ser Ala Ile Val Thr Gly Gly
1 5 10 15
Gly Ser Gly Ile Gly Arg Ala Val Ala Leu Thr Leu Ala Ala Ser Gly
20 25 30
Ala Ala Val Leu Val Thr Asp Leu Asn Glu Glu His Ala Gln Ala Val
35 40 45
Val Ala Glu Ile Glu Ala Ala Gly Gly Lys Ala Ala Ala Leu Ala Gly
50 55 60
Asp Val Thr Asp Pro Ala Phe Gly Glu Ala Ser Val Ala Ala Ala Asn
65 70 75 80
Ala Leu Ala Pro Leu Lys Ile Ala Val Asn Asn Ala Gly Ile Gly Gly
85 90 95
Glu Ala Ala Lys Val Gly Asp Tyr Ser Leu Asp Ser Trp Arg Lys Val
100 105 110
Ile Glu Val Asn Leu Asn Ala Val Phe Tyr Gly Met Gln Pro Gln Leu
115 120 125
Lys Ala Met Ala Ala Asn Gly Gly Gly Ala Ile Val Asn Met Ala Ser
130 135 140
Ile Leu Gly Leu Val Gly Phe Ala Asn Ser Ser Ala Tyr Val Thr Ala
145 150 155 160
Lys His Ala Leu Leu Gly Leu Thr Gln Asn Ala Ala Leu Glu Tyr Ala
165 170 175
Ala Asp Lys Val Arg Val Val Ala Val Gly Pro Gly Phe Ile Arg Thr
180 185 190
Pro Leu Val Glu Ala Asn Leu Ser Ala Glu Ala Leu Ala Phe Leu Glu
195 200 205
Gly Lys His Ala Leu Gly Arg Leu Gly Glu Pro Glu Glu Val Ala Ser
210 215 220
Leu Val Ala Phe Leu Ala Ser Asp Ala Ala Ser Phe Ile Thr Gly Ser
225 230 235 240
Tyr His Leu Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 15
<211> 367
<212> PRT
<213>artificial sequence
<400> 15
Met Thr Lys Ala Val Pro Asp Lys Phe Gln Gly Phe Ala Val Ser Asp
1 5 10 15
Pro Lys Asn Trp Asn Arg Pro Lys Leu Ala Ser Tyr Glu Arg Lys Gln
20 25 30
Ile Asn Pro His Asp Val Val Leu Lys Asn Glu Val Cys Gly Leu Cys
35 40 45
Tyr Ser Asp Ile His Thr Leu Ser Ala Gly Trp Gln Pro Leu Gln Arg
50 55 60
Asp Asn Leu Val Val Gly His Glu Ile Ile Gly Glu Val Ile Ala Val
65 70 75 80
Gly Asp Glu Val Thr Glu Phe Lys Val Gly Asp Arg Val Gly Ile Gly
85 90 95
Ala Ala Ser Ser Ser Cys Arg Ser Cys Gln Arg Cys Asp Ser Asp Asn
100 105 110
Glu Gln Tyr Cys Lys Gln Gly Ala Ala Thr Tyr Asn Ser Lys Asp Val
115 120 125
Arg Ser Asn Asn Tyr Val Thr Gln Gly Gly Tyr Ser Ser His Ser Ile
130 135 140
Ala Asp Glu Lys Phe Val Phe Ala Ile Pro Glu Asp Leu Pro Ser Ser
145 150 155 160
Tyr Gly Ala Pro Leu Met Cys Ala Gly Ile Thr Val Phe Ser Pro Leu
165 170 175
Ile Arg Asn Leu Gly Leu Asp Ala Arg Gly Lys Asn Val Gly Ile Ile
180 185 190
Gly Ile Gly Gly Leu Gly His Leu Ala Leu Gln Phe Ala Asn Ala Met
195 200 205
Gly Ala Asn Val Thr Ala Phe Ser Arg Ser Ser Ser Lys Lys Glu Gln
210 215 220
Ala Met Lys Leu Gly Ala His Asp Phe Val Ala Thr Gly Glu Asp Lys
225 230 235 240
Thr Trp Tyr Lys Asn Tyr Asp Asp His Phe Asp Phe Ile Leu Asn Cys
245 250 255
Ala Ser Gly Ile Asp Gly Leu Asn Leu Ser Glu Tyr Leu Ser Thr Leu
260 265 270
Lys Val Asp Lys Lys Phe Val Ser Val Gly Leu Pro Pro Ser Glu Asp
275 280 285
Lys Phe Glu Val Ser Pro Phe Thr Phe Leu Gln Gln Gly Ala Ser Phe
290 295 300
Gly Ser Ser Leu Leu Gly Ser Lys Thr Glu Val Lys Glu Met Leu Asn
305 310 315 320
Leu Ala Ala Lys His Asn Val Arg Pro Met Ile Glu Glu Val Pro Ile
325 330 335
Ser Glu Glu Asn Cys Ala Lys Ala Leu Asp Arg Cys His Ala Gly Asp
340 345 350
Val Arg Tyr Arg Phe Val Phe Thr Asp Phe Asp Lys Ala Phe Ala
355 360 365
<210> 16
<211> 283
<212> PRT
<213>artificial sequence
<400> 16
Met Ala Lys Asn Phe Ser Asn Val Glu Tyr Pro Ala Pro Pro Pro Ala
1 5 10 15
His Thr Lys Asn Glu Ser Leu Gln Val Leu Asp Leu Phe Lys Leu Asn
20 25 30
Gly Lys Val Ala Ser Ile Thr Gly Ser Ser Ser Gly Ile Gly Tyr Ala
35 40 45
Leu Ala Glu Ala Phe Ala Gln Val Gly Ala Asp Val Ala Ile Trp Tyr
50 55 60
Asn Ser Gln Asp Ala Thr Gly Lys Ala Glu Ala Leu Ala Lys Lys Tyr
65 70 75 80
Gly Val Lys Val Lys Ala Tyr Lys Ala Asn Val Ser Ser Ser Asp Ala
85 90 95
Val Lys Gln Thr Ile Glu Gln Gln Ile Lys Asp Phe Gly His Leu Asp
100 105 110
Ile Val Val Ala Asn Ala Gly Ile Pro Trp Thr Lys Gly Ala Tyr Ile
115 120 125
Asp Gln Asp Asp Asp Lys His Phe Asp Gln Val Ile Asp Val Asp Leu
130 135 140
Lys Gly Val Gly Tyr Val Ala Lys His Ala Gly Arg His Tyr Arg Glu
145 150 155 160
Arg Phe Glu Lys Glu Gly Ile Lys Gly Ala Leu Ile Phe Thr Ala Ser
165 170 175
Val Ser Gly His Ile Val Asn Ile Pro Gln Phe Gln Ala Thr Tyr Asn
180 185 190
Ala Ala Lys Ala Gly Val Arg His Phe Ala Lys Ser Leu Ala Val Glu
195 200 205
Phe Ala Pro Phe Ala Arg Val Asn Ser Val Ser Pro Gly Tyr Ile Asn
210 215 220
Thr Glu Ile Ser Asp Phe Val Pro Gln Glu Thr Gln Asn Lys Trp Trp
225 230 235 240
Ser Leu Val Pro Leu Gly Arg Gly Gly Glu Thr Ala Glu Leu Val Gly
245 250 255
Ala Tyr Leu Phe Leu Ala Ser Asp Ala Gly Ser Tyr Ala Thr Gly Thr
260 265 270
Asp Ile Ile Val Asp Gly Gly Tyr Thr Leu Pro
275 280
<210> 17
<211> 759
<212> DNA
<213>artificial sequence
<400> 17
atgaccgacc gtctgaaaca caaagttgct atcgttaccg gtggtaccat gggtatcggt 60
ctggctatcg ctgacaaata cgttgaagaa ggtgctaaag ttgttatcac cggtcgtcac 120
gctgacgttg gtgaaaaagc tgctaaatct atcggtggta ccgacgttat ccgtttcgtt 180
cagcacgacg tttctgacga agctggttgg accaaactgt tcgacaccac cgaagaaacc 240
ttcggtccgg ttaccaccgt tgttaacaac gctggttctt gcgttctgaa atctgttaaa 300
gacaccacca ccgaagaatg gcgtaaactg ctgtctgtta acctggacgg tgttttcttc 360
ggtacccgtc tgggtatcca gcgtatgaaa aacaaaggtc tgggtgcttc tatcatcaac 420
atgtcttcta tcgaaggtct ggttggtgac ccgtctctgg gtgcttacaa cgcttctaaa 480
ggtgctgttc gtatcatgtc taaatctgct gctctggact gcgctctgaa agactacgac 540
gttcgtgtta acaccgttca cccgggtccg atcaaaaccc agcgtctgga ccagctggaa 600
ggttgggaag aaatgatgtc tcagcgtacc gttaccccga tgggtcgtat cggtgaaccg 660
aacgacatcg cttggatctg cgtttacctg gcttctgacg aatctaaatt cgctaccggt 720
gctgaattcg ttgttgacgg tggttacacc gctcagtaa 759
<210> 18
<211> 759
<212> DNA
<213>artificial sequence
<400> 18
atgaccgacc gtctgaaagg taaagttgct atcgttaccg gtggtaccct gggtatcggt 60
ctggctatcg ctgacaaatt cgttgaagaa ggtgctaaag ttgttatcac cggtcgtcgt 120
gctgacgttg gtgaaaaagc tgctaaatct atcggtggta ccgacgttat ccgtttcgtt 180
cagcacgacg cttctgacga agctggttgg accaaactgt tcgacaccac cgaagaagct 240
ttcggtccgg ttaccaccgt tgttaacaac gctggtatca ccgtttctaa atctgttgaa 300
gacaccacca ccgaagaatg gcgtaaactg ctgtctgtta acctggacgg tgttttcttc 360
ggtacccgtc tgggtatcca gcgtatgaaa aacaaaggtc tgggtgcttc tatcatcaac 420
atgtcttcta tcgaaggtct ggttggtgac ccgaccctgg gtgcttacaa cgcttctaaa 480
ggtgctgttc gtatcatgtc taaatctgct gctctggact gcgctctgaa agactacgac 540
gttcgtgtta acaccgttca cccgggttac atcaaaaccc cgctggttga cgaccacgaa 600
ggtctggaag aaatgatgtc tcagcgtacc aaaaccccga tgggtcacat cggtgaaccg 660
aacgacatcg cttggatctg cgtttacctg gcttctgacg aatctaaatt cgctaccggt 720
gctgaattcg ttgttgacgg tggttacacc gctcagtaa 759
<210> 19
<211> 756
<212> DNA
<213>artificial sequence
<400> 19
atggctcagt acgacgtcgc cgaccggtcc gcgatcgtga ccggaggcgg ctcgggcatc 60
gggcgcgccg tggcgctcac cctggcggcg agcggcgcag ccgtcctcgt caccgacctg 120
aacgaggagc acgcgcaggc cgtcgtggcc gagatcgagg ccgcgggcgg aaaggccgcc 180
gcgctcgccg gcgacgtgac cgaccccgcg ttcggcgagg cgagcgtcgc cgcggcgaac 240
gctctggcac cgctcaagat cgcggtgaac aacgcgggca tcggcggcga ggccgccaca 300
gtcggcgact actcgctcga cagctggcgc aaggtcatcg aggtcaacct caacgccgtg 360
ttctacggga tgcagccgca gctgaaggcc atggccgcga acggcggcgg tgcgatcgtc 420
aacatggcgt ccatcctggg cagcgtcggc ttcgcgaatt cgtcggccta cgtcaccgcc 480
aagcacgcgc tgctcggcct cacgcagaac gccgcgctcg agtacgccgc cgacaaggtg 540
cgcgtcgtcg cggtcggccc cggcttcatc cgcaccccgc tcgtggaggc caacctctcc 600
gccgaggcgc tggcgttcct cgagggcaag cacgccctcg gccgcctggg cgagccggag 660
gaggtcgcct ccctggtcgc gttcctcgcc tccgacgcgg cgagcttcat caccggcagc 720
taccacctgg tggacggcgg ctacaccgct cagtga 756
<210> 20
<211> 756
<212> DNA
<213>artificial sequence
<400> 20
atggctcagt acgacgtcgc cgaccggtcc gcgatcgtga ccggaggcgg ctcgggcatc 60
gggcgcgccg tggcgctcac cctggcggcg agcggcgcag ccgtcctcgt caccgacctg 120
aacgaggagc acgcgcaggc cgtcgtggcc gagatcgagg ccgcgggcgg aaaggccgcc 180
gcgctcgccg gcgacgtgac cgaccccgcg ttcggcgagg cgagcgtcgc cgcggcgaac 240
gctctggcac cgctcaagat cgcggtgaac aacgcgggca tcggcggcga ggccgccaga 300
gtcggcgact actcgctcga cagctggcgc aaggtcatcg aggtcaacct caacgccgtg 360
ttctacggga tgcagccgca gctgaaggcc atggccgcga acggcggcgg tgcgatcgtc 420
aacatggcgt ccatcctggg cagcgtcggc ttcgcgaatt cgtcggccta cgtcaccgcc 480
aagcacgcgc tgctcggcct cacgcagaac gccgcgctcg agtacgccgc cgacaaggtg 540
cgcgtcgtcg cggtcggccc cggcttcatc cgcaccccgc tcgtggaggc caacctctcc 600
gccgaggcgc tggcgttcct cgagggcaag cacgccctcg gccgcctggg cgagccggag 660
gaggtcgcct ccctggtcgc gttcctcgcc tccgacgcgg cgagcttcat caccggcagc 720
taccacctgg tggacggcgg ctacaccgct cagtga 756
<210> 21
<211> 756
<212> DNA
<213>artificial sequence
<400> 21
atggctcagt acgacgtcgc cgaccggtcc gcgatcgtga ccggaggcgg ctcgggcatc 60
gggcgcgccg tggcgctcac cctggcggcg agcggcgcag ccgtcctcgt caccgacctg 120
aacgaggagc acgcgcaggc cgtcgtggcc gagatcgagg ccgcgggcgg aaaggccgcc 180
gcgctcgccg gcgacgtgac cgaccccgcg ttcggcgagg cgagcgtcgc cgcggcgaac 240
gctctggcac cgctcaagat cgcggtgaac aacgcgggca tcggcggcga ggccgccaag 300
gtcggcgact actcgctcga cagctggcgc aaggtcatcg aggtcaacct caacgccgtg 360
ttctacggga tgcagccgca gctgaaggcc atggccgcga acggcggcgg tgcgatcgtc 420
aacatggcgt ccatcctggg cagcgtcggc ttcgcgaatt cgtcggccta cgtcaccgcc 480
aagcacgcgc tgctcggcct cacgcagaac gccgcgctcg agtacgccgc cgacaaggtg 540
cgcgtcgtcg cggtcggccc cggcttcatc cgcaccccgc tcgtggaggc caacctctcc 600
gccgaggcgc tggcgttcct cgagggcaag cacgccctcg gccgcctggg cgagccggag 660
gaggtcgcct ccctggtcgc gttcctcgcc tccgacgcgg cgagcttcat caccggcagc 720
taccacctgg tggacggcgg ctacaccgct cagtga 756
<210> 22
<211> 756
<212> DNA
<213>artificial sequence
<400> 22
atggctcagt acgacgtcgc cgaccggtcc gcgatcgtga ccggaggcgg ctcgggcatc 60
gggcgcgccg tggcgctcac cctggcggcg agcggcgcag ccgtcctcgt caccgacctg 120
aacgaggagc acgcgcaggc cgtcgtggcc gagatcgagg ccgcgggcgg aaaggccgcc 180
gcgctcgccg gcgacgtgac cgaccccgcg ttcggcgagg cgagcgtcgc cgcggcgaac 240
gctctggcac cgctcaagat cgcggtgaac aacgcgggca tcggcggcga ggccgccaca 300
gtcggcgact actcgctcga cagctggcgc aaggtcatcg aggtcaacct caacgccgtg 360
ttctacggga tgcagccgca gctgaaggcc atggccgcga acggcggcgg tgcgatcgtc 420
aacatggcgt ccatcctggg catcgtcggc ttcgcgaatt cgtcggccta cgtcaccgcc 480
aagcacgcgc tgctcggcct cacgcagaac gccgcgctcg agtacgccgc cgacaaggtg 540
cgcgtcgtcg cggtcggccc cggcttcatc cgcaccccgc tcgtggaggc caacctctcc 600
gccgaggcgc tggcgttcct cgagggcaag cacgccctcg gccgcctggg cgagccggag 660
gaggtcgcct ccctggtcgc gttcctcgcc tccgacgcgg cgagcttcat caccggcagc 720
taccacctgg tggacggcgg ctacaccgct cagtga 756
<210> 23
<211> 756
<212> DNA
<213>artificial sequence
<400> 23
atggctcagt acgacgtcgc cgaccggtcc gcgatcgtga ccggaggcgg ctcgggcatc 60
gggcgcgccg tggcgctcac cctggcggcg agcggcgcag ccgtcctcgt caccgacctg 120
aacgaggagc acgcgcaggc cgtcgtggcc gagatcgagg ccgcgggcgg aaaggccgcc 180
gcgctcgccg gcgacgtgac cgaccccgcg ttcggcgagg cgagcgtcgc cgcggcgaac 240
gctctggcac cgctcaagat cgcggtgaac aacgcgggca tcggcggcga ggccgccaca 300
gtcggcgact actcgctcga cagctggcgc aaggtcatcg aggtcaacct caacgccgtg 360
ttctacggga tgcagccgca gctgaaggcc atggccgcga acggcggcgg tgcgatcgtc 420
aacatggcgt ccatcctggg cgtggtcggc ttcgcgaatt cgtcggccta cgtcaccgcc 480
aagcacgcgc tgctcggcct cacgcagaac gccgcgctcg agtacgccgc cgacaaggtg 540
cgcgtcgtcg cggtcggccc cggcttcatc cgcaccccgc tcgtggaggc caacctctcc 600
gccgaggcgc tggcgttcct cgagggcaag cacgccctcg gccgcctggg cgagccggag 660
gaggtcgcct ccctggtcgc gttcctcgcc tccgacgcgg cgagcttcat caccggcagc 720
taccacctgg tggacggcgg ctacaccgct cagtga 756
<210> 24
<211> 756
<212> DNA
<213>artificial sequence
<400> 24
atggctcagt acgacgtcgc cgaccggtcc gcgatcgtga ccggaggcgg ctcgggcatc 60
gggcgcgccg tggcgctcac cctggcggcg agcggcgcag ccgtcctcgt caccgacctg 120
aacgaggagc acgcgcaggc cgtcgtggcc gagatcgagg ccgcgggcgg aaaggccgcc 180
gcgctcgccg gcgacgtgac cgaccccgcg ttcggcgagg cgagcgtcgc cgcggcgaac 240
gctctggcac cgctcaagat cgcggtgaac aacgcgggca tcggcggcga ggccgccaca 300
gtcggcgact actcgctcga cagctggcgc aaggtcatcg aggtcaacct caacgccgtg 360
ttctacggga tgcagccgca gctgaaggcc atggccgcga acggcggcgg tgcgatcgtc 420
aacatggcgt ccatcctggg cctggtcggc ttcgcgaatt cgtcggccta cgtcaccgcc 480
aagcacgcgc tgctcggcct cacgcagaac gccgcgctcg agtacgccgc cgacaaggtg 540
cgcgtcgtcg cggtcggccc cggcttcatc cgcaccccgc tcgtggaggc caacctctcc 600
gccgaggcgc tggcgttcct cgagggcaag cacgccctcg gccgcctggg cgagccggag 660
gaggtcgcct ccctggtcgc gttcctcgcc tccgacgcgg cgagcttcat caccggcagc 720
taccacctgg tggacggcgg ctacaccgct cagtga 756
<210> 25
<211> 756
<212> DNA
<213>artificial sequence
<400> 25
atggctcagt acgacgtcgc cgaccggtcc gcgatcgtga ccggaggcgg ctcgggcatc 60
gggcgcgccg tggcgctcac cctggcggcg agcggcgcag ccgtcctcgt caccgacctg 120
aacgaggagc acgcgcaggc cgtcgtggcc gagatcgagg ccgcgggcgg aaaggccgcc 180
gcgctcgccg gcgacgtgac cgaccccgcg ttcggcgagg cgagcgtcgc cgcggcgaac 240
gctctggcac cgctcaagat cgcggtgaac aacgcgggca tcggcggcga ggccgccaga 300
gtcggcgact actcgctcga cagctggcgc aaggtcatcg aggtcaacct caacgccgtg 360
ttctacggga tgcagccgca gctgaaggcc atggccgcga acggcggcgg tgcgatcgtc 420
aacatggcgt ccatcctggg catcgtcggc ttcgcgaatt cgtcggccta cgtcaccgcc 480
aagcacgcgc tgctcggcct cacgcagaac gccgcgctcg agtacgccgc cgacaaggtg 540
cgcgtcgtcg cggtcggccc cggcttcatc cgcaccccgc tcgtggaggc caacctctcc 600
gccgaggcgc tggcgttcct cgagggcaag cacgccctcg gccgcctggg cgagccggag 660
gaggtcgcct ccctggtcgc gttcctcgcc tccgacgcgg cgagcttcat caccggcagc 720
taccacctgg tggacggcgg ctacaccgct cagtga 756
<210> 26
<211> 756
<212> DNA
<213>artificial sequence
<400> 26
atggctcagt acgacgtcgc cgaccggtcc gcgatcgtga ccggaggcgg ctcgggcatc 60
gggcgcgccg tggcgctcac cctggcggcg agcggcgcag ccgtcctcgt caccgacctg 120
aacgaggagc acgcgcaggc cgtcgtggcc gagatcgagg ccgcgggcgg aaaggccgcc 180
gcgctcgccg gcgacgtgac cgaccccgcg ttcggcgagg cgagcgtcgc cgcggcgaac 240
gctctggcac cgctcaagat cgcggtgaac aacgcgggca tcggcggcga ggccgccaga 300
gtcggcgact actcgctcga cagctggcgc aaggtcatcg aggtcaacct caacgccgtg 360
ttctacggga tgcagccgca gctgaaggcc atggccgcga acggcggcgg tgcgatcgtc 420
aacatggcgt ccatcctggg cgtggtcggc ttcgcgaatt cgtcggccta cgtcaccgcc 480
aagcacgcgc tgctcggcct cacgcagaac gccgcgctcg agtacgccgc cgacaaggtg 540
cgcgtcgtcg cggtcggccc cggcttcatc cgcaccccgc tcgtggaggc caacctctcc 600
gccgaggcgc tggcgttcct cgagggcaag cacgccctcg gccgcctggg cgagccggag 660
gaggtcgcct ccctggtcgc gttcctcgcc tccgacgcgg cgagcttcat caccggcagc 720
taccacctgg tggacggcgg ctacaccgct cagtga 756
<210> 27
<211> 756
<212> DNA
<213>artificial sequence
<400> 27
atggctcagt acgacgtcgc cgaccggtcc gcgatcgtga ccggaggcgg ctcgggcatc 60
gggcgcgccg tggcgctcac cctggcggcg agcggcgcag ccgtcctcgt caccgacctg 120
aacgaggagc acgcgcaggc cgtcgtggcc gagatcgagg ccgcgggcgg aaaggccgcc 180
gcgctcgccg gcgacgtgac cgaccccgcg ttcggcgagg cgagcgtcgc cgcggcgaac 240
gctctggcac cgctcaagat cgcggtgaac aacgcgggca tcggcggcga ggccgccaga 300
gtcggcgact actcgctcga cagctggcgc aaggtcatcg aggtcaacct caacgccgtg 360
ttctacggga tgcagccgca gctgaaggcc atggccgcga acggcggcgg tgcgatcgtc 420
aacatggcgt ccatcctggg cctggtcggc ttcgcgaatt cgtcggccta cgtcaccgcc 480
aagcacgcgc tgctcggcct cacgcagaac gccgcgctcg agtacgccgc cgacaaggtg 540
cgcgtcgtcg cggtcggccc cggcttcatc cgcaccccgc tcgtggaggc caacctctcc 600
gccgaggcgc tggcgttcct cgagggcaag cacgccctcg gccgcctggg cgagccggag 660
gaggtcgcct ccctggtcgc gttcctcgcc tccgacgcgg cgagcttcat caccggcagc 720
taccacctgg tggacggcgg ctacaccgct cagtga 756
<210> 28
<211> 756
<212> DNA
<213>artificial sequence
<400> 28
atggctcagt acgacgtcgc cgaccggtcc gcgatcgtga ccggaggcgg ctcgggcatc 60
gggcgcgccg tggcgctcac cctggcggcg agcggcgcag ccgtcctcgt caccgacctg 120
aacgaggagc acgcgcaggc cgtcgtggcc gagatcgagg ccgcgggcgg aaaggccgcc 180
gcgctcgccg gcgacgtgac cgaccccgcg ttcggcgagg cgagcgtcgc cgcggcgaac 240
gctctggcac cgctcaagat cgcggtgaac aacgcgggca tcggcggcga ggccgccaag 300
gtcggcgact actcgctcga cagctggcgc aaggtcatcg aggtcaacct caacgccgtg 360
ttctacggga tgcagccgca gctgaaggcc atggccgcga acggcggcgg tgcgatcgtc 420
aacatggcgt ccatcctggg catcgtcggc ttcgcgaatt cgtcggccta cgtcaccgcc 480
aagcacgcgc tgctcggcct cacgcagaac gccgcgctcg agtacgccgc cgacaaggtg 540
cgcgtcgtcg cggtcggccc cggcttcatc cgcaccccgc tcgtggaggc caacctctcc 600
gccgaggcgc tggcgttcct cgagggcaag cacgccctcg gccgcctggg cgagccggag 660
gaggtcgcct ccctggtcgc gttcctcgcc tccgacgcgg cgagcttcat caccggcagc 720
taccacctgg tggacggcgg ctacaccgct cagtga 756
<210> 29
<211> 756
<212> DNA
<213>artificial sequence
<400> 29
atggctcagt acgacgtcgc cgaccggtcc gcgatcgtga ccggaggcgg ctcgggcatc 60
gggcgcgccg tggcgctcac cctggcggcg agcggcgcag ccgtcctcgt caccgacctg 120
aacgaggagc acgcgcaggc cgtcgtggcc gagatcgagg ccgcgggcgg aaaggccgcc 180
gcgctcgccg gcgacgtgac cgaccccgcg ttcggcgagg cgagcgtcgc cgcggcgaac 240
gctctggcac cgctcaagat cgcggtgaac aacgcgggca tcggcggcga ggccgccaag 300
gtcggcgact actcgctcga cagctggcgc aaggtcatcg aggtcaacct caacgccgtg 360
ttctacggga tgcagccgca gctgaaggcc atggccgcga acggcggcgg tgcgatcgtc 420
aacatggcgt ccatcctggg cgtggtcggc ttcgcgaatt cgtcggccta cgtcaccgcc 480
aagcacgcgc tgctcggcct cacgcagaac gccgcgctcg agtacgccgc cgacaaggtg 540
cgcgtcgtcg cggtcggccc cggcttcatc cgcaccccgc tcgtggaggc caacctctcc 600
gccgaggcgc tggcgttcct cgagggcaag cacgccctcg gccgcctggg cgagccggag 660
gaggtcgcct ccctggtcgc gttcctcgcc tccgacgcgg cgagcttcat caccggcagc 720
taccacctgg tggacggcgg ctacaccgct cagtga 756
<210> 30
<211> 756
<212> DNA
<213>artificial sequence
<400> 30
atggctcagt acgacgtcgc cgaccggtcc gcgatcgtga ccggaggcgg ctcgggcatc 60
gggcgcgccg tggcgctcac cctggcggcg agcggcgcag ccgtcctcgt caccgacctg 120
aacgaggagc acgcgcaggc cgtcgtggcc gagatcgagg ccgcgggcgg aaaggccgcc 180
gcgctcgccg gcgacgtgac cgaccccgcg ttcggcgagg cgagcgtcgc cgcggcgaac 240
gctctggcac cgctcaagat cgcggtgaac aacgcgggca tcggcggcga ggccgccaag 300
gtcggcgact actcgctcga cagctggcgc aaggtcatcg aggtcaacct caacgccgtg 360
ttctacggga tgcagccgca gctgaaggcc atggccgcga acggcggcgg tgcgatcgtc 420
aacatggcgt ccatcctggg cctggtcggc ttcgcgaatt cgtcggccta cgtcaccgcc 480
aagcacgcgc tgctcggcct cacgcagaac gccgcgctcg agtacgccgc cgacaaggtg 540
cgcgtcgtcg cggtcggccc cggcttcatc cgcaccccgc tcgtggaggc caacctctcc 600
gccgaggcgc tggcgttcct cgagggcaag cacgccctcg gccgcctggg cgagccggag 660
gaggtcgcct ccctggtcgc gttcctcgcc tccgacgcgg cgagcttcat caccggcagc 720
taccacctgg tggacggcgg ctacaccgct cagtga 756
<211> 31
<211> 1104
<212> DNA
<213>artificial sequence
<400> 31
atgaccaaag ctgttccgga caaattccag ggtttcgctg tttctgaccc gaaaaactgg 60
aaccgtccga aactggcttc ttacgaacgt aaacagatca acccgcacga cgttgttctg 120
aaaaacgaag tttgcggtct gtgctactct gacatccaca ccctgtctgc tggttggcag 180
ccgctgcagc gtgacaacct ggttgttggt cacgaaatca tcggtgaagt tatcgctgtt 240
ggtgacgaag ttaccgaatt caaagttggt gaccgtgttg gtatcggtgc tgcttcttct 300
tcttgccgtt cttgccagcg ttgcgactct gacaacgaac agtactgcaa acagggtgct 360
gctacctaca actctaaaga cgttcgttct aacaactacg ttacccaggg tggttactct 420
tctcactcta tcgctgacga aaaattcgtt ttcgctatcc cggaagacct gccgtcttct 480
tacggtgctc cgctgatgtg cgctggtatc accgttttct ctccgctgat ccgtaacctg 540
ggtctggacg ctcgtggtaa aaacgttggt atcatcggta tcggtggtct gggtcacctg 600
gctctgcagt tcgctaacgc tatgggtgct aacgttaccg ctttctctcg ttcttcttct 660
aaaaaagaac aggctatgaa actgggtgct cacgacttcg ttgctaccgg tgaagacaaa 720
acctggtaca aaaactacga cgaccacttc gacttcatcc tgaactgcgc ttctggtatc 780
gacggtctga acctgtctga atacctgtct accctgaaag ttgacaaaaa attcgtttct 840
gttggtctgc cgccgtctga agacaaattc gaagtttctc cgttcacctt cctgcagcag 900
ggtgcttctt tcggttcttc tctgctgggt tctaaaaccg aagttaaaga aatgctgaac 960
ctggctgcta aacacaacgt tcgtccgatg atcgaagaag ttccgatctc tgaagaaaac 1020
tgcgctaaag ctctggaccg ttgccacgct ggtgacgttc gttaccgttt cgttttcacc 1080
gacttcgaca aagcgttcgc ttaa 1104
<210> 32
<211> 852
<212> DNA
<213>artificial sequence
<400> 32
atggctaaaa acttctctaa cgttgaatac ccggctccgc cgccggctca caccaaaaac 60
gaatctctgc aggttctgga cctgttcaaa ctgaacggta aagttgcttc tatcaccggt 120
tcttcttctg gtatcggtta cgctctggct gaagctttcg ctcaggttgg tgctgacgtt 180
gctatctggt acaactctca ggacgctacc ggtaaagctg aagctctggc taaaaaatac 240
ggtgttaaag ttaaagctta caaagctaac gtttcttctt ctgacgctgt taaacagacc 300
atcgaacagc agatcaaaga cttcggtcac ctggacatcg ttgttgctaa cgctggtatc 360
ccgtggacca aaggtgctta catcgaccag gacgacgaca aacacttcga ccaggttatc 420
gacgttgacc tgaaaggtgt tggttacgtt gctaaacacg ctggtcgtca ctaccgtgaa 480
cgtttcgaaa aagaaggtat caaaggtgct ctgatcttca ccgcttctgt ttctggtcac 540
atcgttaaca tcccgcagtt ccaggctacc tacaacgctg ctaaagctgg tgttcgtcac 600
ttcgctaaat ctctggctgt tgaattcgct ccgttcgctc gtgttaactc tgtttctccg 660
ggttacatca acaccgaaat ctctgacttc gttccgcagg aaacccagaa caaatggtgg 720
tctctggttc cgctgggtcg tggtggtgaa accgctgaac tggttggtgc ttacctgttc 780
ctggcttctg acgctggttc ttacgctacc ggtaccgaca tcatcgttga cggtggttac 840
accctgccgt aa 852

Claims (10)

1. a kind of method of enzyme process preparation ticagrelor midbody, which is characterized in that comprising steps of
It uses KRED enzyme as catalyst, is reacted with compound 28 with reducing agent, obtain compound 29:
Wherein, the KRED enzyme is the alcohol dehydrogenase from Leifsonia aquatica ATCC 14665, and its mutation Body；Or the KRED enzyme has the protein sequence as shown in SEQ ID NO:15 or SEQ ID NO:16.
2. the method as described in claim 1, which is characterized in that described derives from Leifsonia aquatica ATCC The mutant of 14665 alcohol dehydrogenase is carried out in site selected from the group below: T100R, T100K, S148I, S148V, S148L, or combinations thereof.
3. the method as described in claim 1, which is characterized in that described derives from Leifsonia aquatica ATCC 14665 alcohol dehydrogenase has the protein sequence as shown in SEQ ID NO:3-SEQ ID NO:14.
4. the method as described in claim 1, which is characterized in that in the reaction, the reducing agent is selected from the group: isopropyl Alcohol, glucose, or combinations thereof.
5. the method as described in claim 1, which is characterized in that the reaction carries out in the presence of co-factor；Preferably, institute The co-factor stated is selected from the group: reducibility coenzyme NAD+, NADP+, or combinations thereof.
6. the method as described in claim 1, which is characterized in that the reaction in the transformation system of pH value 6.5~7.5 into Row.
7. a kind of expression vector, which is characterized in that the expression vector expression includes the DNA sequence as shown in SEQ ID NO:31 Column.
8. a kind of hydro-reduction method, which is characterized in that the described method includes: using KRED enzyme as catalyst；Wherein, described KRED enzyme is the alcohol dehydrogenase and its mutant from Leifsonia aquatica ATCC 14665；Or the KRED Enzyme has the protein sequence as shown in SEQ ID NO:15 or SEQ ID NO:16；
Preferably, the method includes the steps that as follows:
Using compound I as raw material, it is catalyzed with the KRED enzyme, obtains Formula II compound；
Wherein, R1Be selected from the group: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 naphthenic base, replace or not Substituted C6-C10 aryl；
R2Be selected from the group: halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 naphthenic base, replace or not Substituted C6-C10 aryl；
N=1,2,3,4 or 5；
The substitution refers to that one or more hydrogen atoms on group are replaced by substituent group selected from the group below: halogen, C1~C4 alkane Base, C1~C4 halogenated alkyl, hydroxyl, amino, nitro, carboxyl, C1~C4 alkoxy, C1~C4 halogenated alkoxy, cyano.
9. a kind of preparation method of compound 10, which is characterized in that the method includes the steps: (1) with such as claim 1 or 8 Compound 29 is prepared in any method, and compound 10 is prepared with the compound 29 in (2).
10. a kind of preparation method of compound Ticagrelor, which is characterized in that the method includes the steps:
(1) compound 29 is prepared with the method as described in claim 1 or 8 is any, and
(2) Ticagrelor is prepared with the compound 29.
CN201710496576.5A 2017-06-26 2017-06-26 Enzymatic preparation of ticagrelor intermediate Active CN109112166B (en)

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