CN116790569A - Pyruvic acid decarboxylase mutant and application thereof in preparation of alpha-hydroxyketone compound - Google Patents

Pyruvic acid decarboxylase mutant and application thereof in preparation of alpha-hydroxyketone compound Download PDF

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CN116790569A
CN116790569A CN202210361627.4A CN202210361627A CN116790569A CN 116790569 A CN116790569 A CN 116790569A CN 202210361627 A CN202210361627 A CN 202210361627A CN 116790569 A CN116790569 A CN 116790569A
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CN116790569B (en
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周硕
毛旭丹
龙柳妃
劳淑华
李渤
李树学
崔宏鹏
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CHIFENG ARKER PHARMACEUTICAL TECHNOLOGY CO LTD
Hangzhou Meiyi Biotechnology Co ltd
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Abstract

The application discloses a pyruvate decarboxylase mutant and application thereof in preparing alpha-hydroxyketone compounds, wherein the pyruvate decarboxylase mutant is prepared by the following steps of: 1, wherein the pyruvate decarboxylase mutant can be applied to preparing alpha-hydroxyketone compounds, the substrate conversion rate can reach 99.7%, and the e.e value of a target product can reach 99.8%, compared with a target product with the amino acid sequence shown as SEQ ID NO:1, and the pyruvate decarboxylase mutant has the advantages of high catalytic activity, better optical selectivity, stronger substrate tolerance and ideal thermal stability.

Description

Pyruvic acid decarboxylase mutant and application thereof in preparation of alpha-hydroxyketone compound
Technical Field
The application relates to the fields of enzyme engineering and biopharmaceuticals, in particular to a pyruvic acid decarboxylase mutant and application thereof in preparing alpha-hydroxyketone compounds.
Background
Pyruvate decarboxylase (pyruvate decarboxylase, PDC), which is an intracellular enzyme widely occurring in animals, plants and microorganisms, is a thiamine pyrophosphate (TPP) dependent non-oxidase. The PDC can be applied to the synthesis of alpha-hydroxyketone compounds.
The alpha-hydroxy ketone compound is a compound with important application value, can be used as a natural product and a medical intermediate with biological activity, can be used as a photoinitiator in ultraviolet light curing coating, and can be derived and converted into heterocyclic compounds. The method for synthesizing the alpha-hydroxyketone compound by adopting PDC catalysis has the advantages of mild reaction conditions, low raw materials and high optical purity of the product, and has great economic value and environmental protection significance. However, the catalytic synthesis of the alpha-hydroxyketone compound by using the wild PDC has the defects of low catalytic activity, poor enzyme stability and insufficient optical selectivity, thereby limiting the industrial application of the PDC in the technical field of the synthesis of the alpha-hydroxyketone compound.
Therefore, how to modify the wild PDC to improve the catalytic activity and the stability of the PDC is of great significance to the development of the alpha-hydroxy ketone compounds prepared by the PDC enzymatic method.
Disclosure of Invention
The application provides a pyruvate decarboxylase mutant and application thereof in preparing alpha-hydroxyketone compounds, so as to solve the problems of low catalytic activity, poor enzyme stability and insufficient optical selectivity in the prior art of catalyzing and synthesizing the alpha-hydroxyketone compounds by using wild pyruvate decarboxylase.
In a first aspect, the application provides a pyruvate decarboxylase mutant having an amino acid sequence identical to SEQ ID NO:1, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similarity, and the amino acid sequence of the pyruvate decarboxylase mutant is represented by SEQ ID NO:1, wherein the amino acid sequence is obtained by one or more point mutations in the amino acid sequence shown in 1; the pyruvate decarboxylase mutant has pyruvate decarboxylase activity.
Further, the amino acid at which the point mutation occurs comprises SEQ ID NO:1, at least one of amino acid T at position 7, amino acid T at position 8, amino acid L at position 38, amino acid N at position 169, amino acid a at position 246, amino acid G at position 294, amino acid W at position 392, amino acid V at position 450, amino acid Q at position 452, amino acid I at position 472, amino acid M at position 475, amino acid I at position 476, amino acid V at position 549, amino acid W at position 551, amino acid K at position 553, or amino acid V at position 555.
Further, the means for generating the point mutation comprises at least one of T7R, T8W, L38M, N Y, A246M, G294K, W392A, V450E, Q452G, I472M, M475K, I476L, V549Y, W551D, K553Y or V555P.
Further, the point mutation occurs in any one of the following ways:
(1)W392I；
(2)I472M；
(3)W551D；
(4)G294K；
(5)I476L；
(6)V555P；
(7)M475K；
(8) W551D and M475K;
(9) V549Y and W392A;
(10) T8W and a246M;
(11) N169Y and V450E;
(12) M475K, L M and W551D;
(13) T7R, L M and Q452G;
(14) T7R, K Y and M475K; or (b)
(15) T7R, L M, W551D and M475K.
Further, the amino acid sequence of the pyruvate decarboxylase mutant is selected from the group consisting of SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13. SEQ ID NO: 15. SEQ ID NO: 17. SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO: 23. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO:29 or SEQ ID NO:31, and a polypeptide comprising the amino acid sequence shown in any one of seq id no.
In a second aspect, the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding a pyruvate decarboxylase mutant according to any of the first aspects.
Further, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 14. SEQ ID NO: 16. SEQ ID NO: 18. SEQ ID NO: 20. SEQ ID NO: 22. SEQ ID NO: 24. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO:30 or SEQ ID NO:32, or a nucleotide sequence as set forth in any one of seq id no.
In a third aspect, the present application provides a recombinant expression vector comprising a vector, and a nucleic acid molecule as in any one of the second aspects; the vector is selected from a plasmid, cosmid, phage or viral vector.
In a fourth aspect, the present application provides a recombinant expression transformant comprising a host, and the nucleic acid molecule according to any one of the second aspects, or the recombinant expression vector according to the third aspect, introduced into the host; the host is selected from eukaryotes or prokaryotes.
In a fifth aspect, the present application provides a method for producing a pyruvate decarboxylase mutant by culturing the recombinant expression transformant described in the fourth aspect, and obtaining the pyruvate decarboxylase mutant from the culture.
In a sixth aspect, the present application provides a method for preparing a chiral α -hydroxyketone compound, wherein the first compound and the second compound are contacted and reacted to form the α -hydroxyketone compound, using the pyruvate decarboxylase mutant as described in any one of the first aspect or the pyruvate decarboxylase mutant prepared by the preparation method as described in the fifth aspect as a catalyst; wherein the first compound has a structure represented by the following general formula (I):
In the general formula (I), R 1 Selected from hydrogen atoms or hydroxyl groups, and X is selected from hydrogen atoms or monovalent metal ions.
The second compound has a structure represented by the following general formula (II):
in the general formula (II), R 2 Selected from aryl or heteroaryl groups.
Further, the first compound is selected from at least one of pyruvic acid, hydroxy pyruvic acid or sodium pyruvic acid, and the second compound is selected from benzaldehyde.
The application provides a pyruvic acid decarboxylase mutant and application thereof in preparing alpha-hydroxyketone compounds, and the pyruvic acid decarboxylase mutant has the following technical effects:
in the application, the pyruvate decarboxylase mutant is shown in SEQ ID NO:1, wherein the pyruvate decarboxylase mutant has pyruvate decarboxylase activity, and the pyruvate decarboxylase mutant can be applied to the preparation of alpha-hydroxyketone compounds, and the catalytic synthesis of the compounds with the structure shown in the formula (IV) is taken as an example, the conversion rate of substrate benzaldehyde can reach 99.7%, and the e.e value of a target product can reach 99.8%, compared with the compounds with the structure shown in SEQ ID NO:1, and the pyruvate decarboxylase mutant has the advantages of high catalytic activity and better optical selectivity. Furthermore, the substrate tolerance test and the thermal stability test show that the tolerance performance and the thermal stability performance of the pyruvate decarboxylase mutant to the substrate are obviously superior to those of the wild PDC.
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The technical solution of the present application and its advantageous effects will be made apparent by the following detailed description of the specific embodiments of the present application with reference to the accompanying drawings.
FIG. 1 is an HPLC chart of a reaction solution obtained by reacting a reaction system including a pyruvate decarboxylase mutant M3 for 2 hours in Experimental example 1;
FIG. 2 is an HPLC chart of a reaction solution obtained by reacting a reaction system including a pyruvate decarboxylase mutant M15 for 2 hours in Experimental example 1;
FIG. 3 is a HPLC chart of a reaction solution obtained by reacting a reaction system including a pyruvate decarboxylase mutant M3 of Experimental example 2 for 30 min;
FIG. 4 is a HPLC chart of a reaction solution obtained by reacting a reaction system including a pyruvate decarboxylase mutant M3 for 24 hours in Experimental example 2;
FIG. 5 is a HPLC chart of a reaction solution obtained by reacting a reaction system including a pyruvate decarboxylase mutant M15 of Experimental example 2 for 30 min;
FIG. 6 is a HPLC chart of a reaction solution obtained by reacting a reaction system including a pyruvate decarboxylase mutant M15 for 24 hours in Experimental example 2;
FIG. 7 is an HPLC chart of a reaction solution obtained by feeding benzaldehyde for 1h to a pre-reaction system comprising a pyruvate decarboxylase mutant M3 in Experimental example 3;
FIG. 8 is an HPLC chart of a reaction solution obtained by feeding benzaldehyde for 24 hours to a pre-reaction system comprising a pyruvate decarboxylase mutant M3 in experimental example 3;
FIG. 9 is an HPLC chart of a reaction solution obtained by feeding benzaldehyde for 1h to a pre-reaction system comprising pyruvic acid decarboxylase mutant M15 in experimental example 3;
FIG. 10 is an HPLC chart of a reaction solution obtained by feeding benzaldehyde for 24 hours to a pre-reaction system containing pyruvic acid decarboxylase mutant M15 in experimental example 3.
Detailed Description
The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to fall within the scope of the application.
The embodiment of the application provides a pyruvate decarboxylase mutant and application thereof in preparing alpha-hydroxyketone compounds, wherein the amino acid sequence of the pyruvate decarboxylase mutant is identical to that of SEQ ID NO:1, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similarity, and the amino acid sequence of the pyruvate decarboxylase mutant is defined by SEQ ID NO:1, and the pyruvate decarboxylase mutant has the activity of pyruvate decarboxylase.
As used herein, "similarity" refers to the relatedness between two amino acid sequences or between two nucleotide sequences, for example: amino acid sequence of pyruvate decarboxylase mutant and SEQ ID NO:1, and a correlation between amino acid sequences shown in 1. In embodiments of the application, at least 80% similarity is understood to be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity, the number of corresponding similarities being an integer; it is further understood that 80.1%, 81.2%, 82.3%, 83.4%, 84.5%, 85.6%, 86.7%, 87.8%, 88.9%, 89.8%, 90.3%, 91.7%, 92.2%, 93.5%, 94.8%, 95.9%, 96.6%, 97.5%, 98.4% or 99.9%, but less than 100% sequence similarity, the number of corresponding similarity being in decimal.
As used herein, "point mutation" refers to a mutation in SEQ ID NO:1, and substitution, deletion or insertion of an amino acid at a specific site in the amino acid sequence shown in fig. 1.
Further, the amino acids with point mutations include SEQ ID NO:1, at least one of amino acid T at position 7, amino acid T at position 8, amino acid L at position 38, amino acid N at position 169, amino acid a at position 246, amino acid G at position 294, amino acid W at position 392, amino acid V at position 450, amino acid Q at position 452, amino acid I at position 472, amino acid M at position 475, amino acid I at position 476, amino acid V at position 549, amino acid W at position 551, amino acid K at position 553, or amino acid V at position 555.
In some embodiments of the application, the manner in which the point mutation occurs comprises at least one of T7R, T W, L38M, N169Y, A246M, G294K, W392A, V450E, Q452G, I472M, M475K, I476L, V549Y, W551D, K553Y or V555P.
As used herein, "amino acid" is represented by a single letter or three letter code, having the following meaning: a: ala (alanine); r: arg (arginine); n: asn (asparagine); d: asp (aspartic acid); c: cys (cysteine); q: gln (glutamine); e: glu (glutamic acid); g: gly (glycine); h: his (histidine); i: ile (isoleucine); l: leu (leucine); k: lys (lysine); m: met (methionine); f: phe (phenylalanine); p: pro (proline); s: ser (serine); t: thr (threonine); w: trp (tryptophan); y: tyr (tyrosine); v: val (valine).
For the point mutation pattern of amino acid substitution, the nomenclature method is: original amino acid, site of original amino acid, substituted amino acid, for example: W551D represents the amino acid sequence set forth in SEQ ID NO:1, substituting aspartic acid for original tryptophan at 551 th site in the amino acid sequence shown in the formula 1; M475K is represented in SEQ ID NO:1, and substituting lysine for original lysine at 475 th site in the amino acid sequence shown in figure 1.
In some embodiments of the application, the amino acid sequence of the pyruvate decarboxylase mutant is selected from the group consisting of SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13. SEQ ID NO: 15. SEQ ID NO: 17. SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO: 23. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO:29 or SEQ ID NO:31, and a polypeptide comprising the amino acid sequence shown in any one of seq id no.
The embodiment of the application also provides a nucleic acid molecule, which comprises a nucleotide sequence for encoding the pyruvate decarboxylase mutant according to any one of the embodiments of the application.
As used herein, "nucleic acid molecule" refers to a biological macromolecular compound polymerized from a plurality of nucleotides, which may be any one of a deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or oligonucleotide fragment produced by Polymerase Chain Reaction (PCR) or by in vitro translation, and a fragment produced by any one or more of ligation, cleavage, endonuclease action or exonuclease action, and may be single-stranded or double-stranded. In embodiments of the application, nucleic acid molecules include, but are not limited to, polynucleotides encoding pyruvate decarboxylase mutants.
In some embodiments of the application, the nucleotide sequence encoding the pyruvate decarboxylase mutant is selected from the group consisting of SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 14. SEQ ID NO: 16. SEQ ID NO: 18. SEQ ID NO: 20. SEQ ID NO: 22. SEQ ID NO: 24. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO:30 or SEQ ID NO:32, or a nucleotide sequence as set forth in any one of seq id no.
For ease of understanding, table 1 below shows specific information on pyruvate decarboxylase mutants involved in the examples of the present application,
the pyruvate decarboxylase mutant has a relative amino acid sequence of SEQ ID NO:1, the pyruvate decarboxylase mutants provided in table 1 are by way of example only:
table 1 list of specific information on pyruvate decarboxylase mutants involved in the examples of the present application
The embodiment of the application also provides a recombinant expression vector, which comprises the vector and the nucleic acid molecule according to any one of the embodiments of the application.
As used herein, a "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid, and may be, for example, a plasmid, virus, cosmid, phage, or the like. In one embodiment of the present application, pET24a into which no foreign gene is inserted may be used as a vector.
As used herein, a "recombinant expression vector" refers to a DNA construct comprising a nucleic acid molecule operably linked to suitable control sequences capable of effecting the expression of the nucleic acid molecule in a suitable host. In the examples of the present application, the recombinant expression vector refers to a DNA construct formed by inserting a nucleic acid molecule encoding a pyruvate decarboxylase mutant into a vector using molecular biological techniques.
The embodiment of the application also provides a recombinant expression transformant, which comprises a host and the nucleic acid molecule or the recombinant expression vector according to any one of the embodiment of the application introduced into the host.
As used herein, a "recombinant expression transformant" refers to a host that has been subjected to exogenous genetic material (e.g., plasmid DNA) to alter genetic characteristics. In an embodiment of the application, the engineering strain that receives the exogenous genetic material (nucleic acid molecule encoding a pyruvate decarboxylase mutant or recombinant expression vector) belongs to a recombinant expression transformant. In one embodiment of the application, the T1 recombinant engineering bacteria to the T15 recombinant engineering bacteria belong to recombinant expression transformants.
As used herein, "host" refers to a class of organisms used to express exogenous genes to produce proteins, and the host may be, for example, eukaryotes, prokaryotes, viruses, etc., wherein eukaryotes as hosts include, but are not limited to, mammalian cells, yeast, fungi, insect cells, and plant cells, and prokaryotes as hosts include, but are not limited to, bacillus, clostridium, lactobacillus, streptomyces, staphylococcus, escherichia, pseudomonas, and paenibacillus. In one embodiment of the application, the host for expressing the pyruvate decarboxylase mutant is E.coli BL21 (DE 3).
The embodiment of the application also provides a preparation method of the pyruvate decarboxylase mutant, which specifically comprises the following steps: by culturing the recombinant expression transformant according to any one of the embodiments of the present application, and obtaining a pyruvate decarboxylase mutant from the culture.
The embodiment of the application also provides a preparation method of the alpha-hydroxy ketone compound, which comprises the following steps: the pyruvate decarboxylase mutant or the pyruvate decarboxylase mutant prepared by any one of the preparation methods disclosed by the embodiment of the application is used as a catalyst, and the first compound and the second compound are in contact reaction to generate the alpha-hydroxyketone compound, wherein the first compound has a structure shown as the following general formula (I):
In the general formula (I), R 1 Selected from hydrogen atoms or hydroxyl groups, and X is selected from hydrogen atoms or monovalent metal ions.
The second compound has a structure represented by the following general formula (II):
in the general formula (II), R 2 Selected from aryl or heteroaryl groups.
As used herein, "aryl" includes both unsubstituted aryl groups and aryl groups wherein one or more hydrogen atoms are optionally substituted with other groups, such as halogen atoms or alkyl groups, allowing multiple degrees of substitution to occur; "unsubstituted aryl" refers to an aromatic group containing only carbon atoms in the aromatic ring, including but not limited to phenyl, 1-naphthyl, 2-naphthyl, or biphenyl.
As used herein, "heteroaryl" means that one or more carbon atoms in the aryl group are independently replaced by one or more heteroatoms (e.g., N, O, P and/or S), e.g., heteroaryl groups have 3 to 20 carbon atoms, and as heteroaryl groups have 5 to 15 carbon atoms, and as heteroaryl groups have 5 to 9 carbon atoms, heteroaryl groups may be unsubstituted or have one or more hydrogen atoms thereon optionally replaced by other groups, e.g., alkyl groups, halogens, and the like, allowing multiple degrees of substitution.
It is understood that the pyruvate decarboxylase mutant used for preparing the alpha-hydroxyketone compound may be a culture (including a culture medium) of a recombinant engineering bacterium carrying a gene encoding the pyruvate decarboxylase mutant, or may be a somatic cell, a somatic cell extract, a somatic cell fragment or a purified pyruvate decarboxylase mutant obtained by separating and purifying the culture.
In some embodiments of the application, in a reaction system in which the pyruvate decarboxylase mutant catalyzes a contact reaction of a first compound and a second compound to produce an α -hydroxyketone compound, the pyruvate decarboxylase mutant: a first compound: the mass ratio of the second compound is 1: (0.7-10): (4.5-12). If the addition amount of the pyruvate decarboxylase mutant is too small, the catalytic reaction effect on the first compound and the second compound is limited, so that the phenomenon of excessive substrates occurs; if the addition amount of the pyruvate decarboxylase mutant is too large, the pyruvate decarboxylase mutant is wasted, the production cost is increased, and the difficulty is increased for the separation and purification of the subsequent products.
Further, the reaction system also comprises thiamine pyrophosphate and Mg 2+ Wherein thiamine pyrophosphate is used as a coenzyme for the decarboxylation reaction of the first compound, mg 2+ For increasing the reactivity of the enzyme, wherein the catalytic reaction is represented by the following formula (III):
in some embodiments of the application, the pH of the reaction system is 5.6 to 7.0 and the reaction temperature is 26 ℃ to 35 ℃. The catalytic reaction may be carried out under shaking or stirring conditions, and the reaction time is, for example, based on a substrate remaining amount of less than 5%; after the catalytic reaction is completed, the α -hydroxy ketone compound may be extracted according to a separation and purification method common in the art, including but not limited to at least one of filtration, centrifugation, precipitation, or drying.
The technical solutions in the embodiments of the present application will be clearly and completely described below. It will be apparent that the described embodiments are only some, but not all, embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to fall within the scope of the application. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present application. The preferred methods and materials described herein are illustrative only and should not be construed as limiting the application.
Unless otherwise indicated, the starting materials and reagents used in the following examples are commercially available or may be prepared by methods known in the art.
1. Description of the Medium in the examples of the application
(1) LB medium
Each 100mL of LB liquid medium comprises: 1.0g of peptone, 0.5g of yeast powder, and 1.0g of NaCl;
for LB solid culture medium, 20g/L agar is added on the basis of the formula of LB liquid culture medium;
for LB medium containing kanamycin resistance, the total concentration of kanamycin was 50. Mu.g/mL.
(2) Self-induction culture medium
120g of yeast powder, 32.25g of peptone and 0.75g of magnesium sulfate (MgSO were weighed out separately 4 ) 16.5g of ammonium sulphate ((NH 4) 2 SO 4 ) 32.5g of monopotassium phosphate (KH) 2 PO 4 ) 35.5g of disodium hydrogen phosphate (Na 2 HPO 4 ) 2.5g of glucose and 10g of alpha-lactose, and then all the weighed components are added into a pulverizer to be sufficiently ground into powder, so as to obtain a powdery self-induction culture medium. 50g of the powdered self-induction medium was dissolved in 1L of deionized water, and after thorough mixing, the pH was adjusted to 7.0, and then sterilized at 121℃for 30min.
2. The description of plasmids and competent cells in the examples of the application is given in Table 2 below:
TABLE 2 description of plasmids and competent cells in the examples of the application
3. Gene fragments and reagents involved in the examples of the present application were described:
the gene fragment related in the embodiment of the application comprises a primer, a sequence shown as SEQ ID NO:2, and the like are synthesized by the division of biological engineering (Shanghai).
Restriction enzymes (e.g., bamH I, nde I and Dpn I), T4 ligase, KOD high-fidelity enzyme kit, gel recovery kit, 10 XT 4 ligase Buffer (Buffer), double distilled water (ddH) 2 Molecular reagents such as O) were purchased from Takara Bio Inc.
The technical scheme and advantageous effects of the present application are further described below with reference to examples.
Example 1: construction of recombinant expression vector pET24a-ZM
Using gene mining techniques, mining a pyruvate decarboxylase derived from zymomonas mobilis (Zymomonas mobilis) from the NCBI database, the pyruvate decarboxylase having an amino acid sequence as set forth in SEQ ID NO:1, NCBI accession number WP_014849477.1. Codon optimization is carried out according to E.coli codon preference, and the coding SEQ ID NO is synthesized by a total gene synthesis method: 1, which has the amino acid sequence shown in SEQ ID NO: 2.
The construction method of the recombinant expression vector pET24a-ZM comprises the following steps of:
s1.1, SEQ ID NO:2, respectively adding enzyme cutting sites BamH I and Nde I at two ends of the nucleotide sequence shown in the formula 2, artificially synthesizing a gene fragment, performing double enzyme cutting on the synthesized gene fragment by adopting BamH I and Nde I restriction enzymes, and recovering the target gene fragment by gel after 1% agarose gel electrophoresis detection of complete enzyme cutting, wherein the operation of recovering the target gene fragment after double enzyme cutting is implemented according to the operation instruction of a gel recovery kit;
s1.2, performing double enzyme digestion on the pET24a plasmid by adopting BamH I and Nde I restriction enzymes, and recovering a carrier skeleton by using 1% agarose gel electrophoresis after detecting that enzyme digestion is complete, wherein the operation of recovering the carrier skeleton after double enzyme digestion is implemented according to the operation instruction of a gel recovery kit;
s1.3, mixing the target gene fragment obtained in the step S1.1 with the vector skeleton obtained in the step S1.2, connecting overnight AT 16 ℃ under the action of T4 ligase, then converting the connection product into DH5a competent cells, picking up monoclonal son for sequencing verification, extracting recombinant plasmid with correct sequencing, obtaining recombinant expression vector containing aminotransferase encoding gene, and naming pET24a-AT, wherein the recombination system is 20 mu L, specifically: 2. Mu.L of 10 XT 4 ligase Buffer (Buffer), 5. Mu.L of the target gene fragment, 5. Mu.L of the vector backbone, 2. Mu.L of T4 ligase and 6. Mu.L of double distilled water (ddH) 2 O)。
EXAMPLE 2 construction of directed mutant library of recombinant engineering bacteria
The pET24a-AT constructed in example 1 is used as a DNA template by adopting a site-directed mutagenesis strategy, a point mutation primer is designed by utilizing Oligo7 software according to the amino acid site to be mutated, and mutation is introduced into the 5' end of the upstream and downstream mutation primer in a manner of inserting, replacing or deleting a base, wherein the mutation site is shown in Table 1. It should be noted that, according to the manner of introducing mutation sites, the skilled person can obtain the nucleotide sequence of the mutation primer in combination with the basic principle of primer design. To construct W392I, the nucleotide sequence of the upstream mutation primer is shown as SEQ ID NO:33, the nucleotide sequence of the downstream mutation primer is shown in SEQ ID NO:34, fifteen sets of mutant primer pairs were obtained by a total design to introduce fifteen mutant forms, respectively, as shown in table 1.
The recombinant expression vector pET24a-AT constructed in example 1 was selected as a template, fifteen sets of mutation primer pairs were used as PCR primers, respectively, and inverse PCR was performed using a KOD high-fidelity enzyme kit, thereby obtaining fifteen mutation sequences. Wherein, the reaction procedure of inverse PCR is: pre-denaturation at 95℃for 3min; denaturation at 98℃for 30s, annealing at 55℃for 30s, extension at 68℃for 3min,28 cycles; extending at 72℃for 5min.
Fifteen mutant sequences are treated by Dpn I restriction endonucleases respectively, enzyme digestion products are connected by T4 ligase and then are converted into escherichia coli BL21 (DE 3) competent, then LB resistance plates containing kanamycin are coated, the plates are placed in 37 ℃ for inversion culture for 18 hours, single colony is selected and transferred into LB liquid culture medium containing kanamycin, the culture solution is selected for sample feeding and sequencing, and clones with correct sequencing are preserved for standby, so that recombinant engineering bacteria taking escherichia coli as hosts are obtained, and recombinant engineering bacteria T1 to T15 respectively used for expressing pyruvate decarboxylase mutants M1 to M15 are obtained.
Example 3 inducible expression and post-treatment of recombinant engineering bacteria
The recombinant engineering bacteria obtained in the example 2 are inoculated into LB liquid medium containing 50 mug/mL kanamycin, and cultured at 37 ℃ and 180r/min until the OD600 is 0.6-0.8, thus obtaining seed bacterial liquid. Seed bacterial liquid is inoculated to fresh self-induction culture medium containing kanamycin with the final concentration of 50 mug/mL at the volume concentration of 1%, and the culture medium is obtained after the seed bacterial liquid is placed at 30 ℃ for 18 hours. Centrifuging the culture solution at 25deg.C and 8000r/min for 10min, discarding supernatant to collect precipitate, washing the precipitate with PB buffer solution with pH of 7.0 for several times, and collecting wet thallus for use.
The wet cells thus obtained were resuspended in ultrapure water to obtain a bacterial liquid having a cell concentration (mg/L) of 20%. The bacterial liquid is treated by adopting an ultrasonic crushing method or a high-pressure homogenizing crushing method, and the crushing conditions can be selected according to actual needs. The working parameters of the example ultrasonic disruption method are: crushing for 1s; suspending for 2s; crushing for 10min under the power of 180W. The working parameters of the example high pressure homogeneous crushing method are: the crushing was carried out twice at 50HZ and 800 bar.
After the bacterial liquid is crushed, the bacterial liquid is centrifuged for 10min to 15min at the temperature of 4 ℃ and 12000r/min to remove cell fragments and macromolecular impurities, and the supernatant is collected and stored at the temperature of-20 ℃ and 4 ℃ for standby, and is the enzyme liquid containing the pyruvate decarboxylase mutant.
Comparative example 1
The comparative example provides a pyruvate decarboxylase, the amino acid sequence of which is shown in SEQ ID NO:1, encodes a polypeptide as set forth in SEQ ID NO:1 is shown in the specification, and the nucleotide sequence of the amino acid sequence shown in SEQ ID NO: 2.
The recombinant expression vector pET24a-ZM is transformed into escherichia coli BL21 (DE 3) to be competent, then a LB resistance plate containing kanamycin is coated, the plate is placed in 37 ℃ for inversion culture for 18 hours, single colony is selected and transferred into LB liquid culture medium containing kanamycin, the culture solution is selected for sample feeding and sequencing, and the clone with correct sequencing is preserved for standby, so that recombinant engineering bacterium T0 is obtained.
Recombinant engineering bacteria T0 are subjected to induced expression and post-treatment according to the method of the example 3, and the obtained enzyme solution is stored at-20 ℃ and 4 ℃ for later use.
Experimental example 1 preliminary comparison of catalytic reactivity and optical Selectivity of pyruvate decarboxylase mutants M1 to M15
A100 mL flask was taken, and 40mL of PB buffer (pH 6.5) having a concentration of 0.1mol/L, 10mL of dimethyl sulfoxide (DMSO), and 50 were sequentially added theretomu.L of thiamine pyrophosphate (TPP) at a concentration of 50Mg/mL and 50 mu.L of Mg at a concentration of 2mol/L 2+ Buffer solution, gently stir well, then add substrate: 500. Mu.L of benzaldehyde and 0.6g of sodium pyruvate, and after the substrate was sufficiently dissolved, the pH of the system was adjusted to 6.5 using 10% NaOH to obtain a mother liquor.
A total of sixteen sets of living systems (corresponding to the living system comprising pyruvate decarboxylase mutant M1 to the living system comprising pyruvate decarboxylase mutant M15, and the living system comprising pyruvate decarboxylase of comparative example 1, respectively) were prepared by mixing 1mL of the mother liquor with 5. Mu.L of the enzyme solution comprising the single pyruvate decarboxylase mutant (prepared in example 3). Placing each group of living detection systems in a shaking table at 30 ℃ and 180r/min for reaction for 2 hours, and detecting and analyzing a reaction liquid by using a high performance liquid chromatography (High Performance Liquid Chromatography, HPLC) method after the reaction is finished, wherein the structural formula of a target product in the reaction liquid is shown as the following formula (IV):
Wherein, the instrument model of HPLC is Shimadzu LC-16 detector, and the working condition of HPLC is as follows:
(1) Preparing a sample injection liquid: mixing 500. Mu.L of the reaction solution with 500. Mu.L of paraxylene, and centrifuging to collect the supernatant; taking 10 mu L of supernatant, adding 990 mu L of mobile phase into the supernatant, shaking and mixing uniformly, and then sampling with the sample feeding amount of 10 mu L each time;
(2) Chromatographic column: macroxyloid OD-H column, 250 x 4.6mm,5 μm.
(3) Preparation of mobile phase: anhydrous n-hexane and anhydrous isopropanol were mixed according to n-hexane: the volume ratio of the isopropanol is 9:1 are mixed and prepared.
(4) Flow rate: 1mL/min.
(5) Analysis time: 20min.
(6) Column temperature: 30 ℃.
The substrate conversion (%) was calculated from the decrease amount of the substrate compound, and the calculation formula of the substrate conversion (%) was as follows:
in formula (III), A0 is the substrate peak area and A1 is the product peak area.
As an example, fig. 1 shows an HPLC profile of a reaction solution obtained by reacting a reaction system containing the pyruvate decarboxylase mutant M3 for 2 hours, and fig. 2 shows an HPLC profile of a reaction product obtained by reacting a reaction system containing the pyruvate decarboxylase mutant M15 for 2 hours.
The conversion of the substrates by the respective pyruvate decarboxylase mutants (M1 to M15) and the pyruvate decarboxylase of comparative example 1, and the optical purity of the corresponding target products produced are detailed in Table 3 below:
TABLE 3 preliminary Activity measurement data for pyruvate decarboxylase mutants M1 to M15 and pyruvate decarboxylase of comparative example 1
As can be seen from table 3, the pyruvate decarboxylase mutants M1 to M15 are mutants obtained by making one or more site mutations based on the pyruvate decarboxylase of comparative example 1, the conversion rate of the substrates by M1 to M15 is significantly improved as compared with the pyruvate decarboxylase of comparative example 1, and the optical purity of the target product generated by the corresponding reaction system is also significantly improved. Taking the pyruvate decarboxylase mutant M15 as an example, the conversion rate of the M15 to the substrate can reach 24.32%, which is 12 times of the conversion rate of the pyruvate decarboxylase of the comparative example 1 to the substrate; in addition, the optical purity of the target product produced by the reaction system containing M15 can reach 99.8%, which is 1.1 times the optical purity of the target product produced by the reaction system containing pyruvate decarboxylase in comparative example 1.
In addition, for the pyruvate decarboxylase mutant obtained by adopting a single site mutation mode, the substrate conversion rate of the pyruvate decarboxylase mutant obtained by adopting a W551D point mutation mode is the highest, and then M475K is adopted; for the pyruvate decarboxylase mutant obtained by the multi-site combined mutation mode, the comprehensive catalytic performance of the pyruvate decarboxylase mutant obtained by the T7R, L38M, W551D and M475K combined mutation mode is optimal.
Experimental example 2 counterscreen comparison of the conversion of substrates by pyruvate decarboxylase mutants M1 to M15
Providing a clean three-neck flask with a magnetic stirring rotor, sequentially adding 55g of pure water and 6.4g of sodium pyruvate into the three-neck flask, placing the three-neck flask containing the pure water and the sodium pyruvate into a constant-temperature magnetic stirring water bath kettle at 30 ℃, starting stirring, and sequentially adding 6g of benzaldehyde and 100 mu L of Mg with the concentration of 2mol/L into the three-neck flask 2+ Buffer and 100. Mu.L of thiamine pyrophosphate (TPP) at a concentration of 50mg/mL to obtain a mixed system, pH of the mixed system was adjusted to 5.95.+ -. 0.2 with 10% (mass/volume, w/v) NaOH, and then 7.0mL of an enzyme solution containing a single pyruvate decarboxylase mutant (produced in example 3) or a pyruvate decarboxylase enzyme solution of comparative example 1 was continuously added to a three-necked flask to obtain a reaction system, and pH of the reaction system was adjusted to 6.2.+ -. 0.2 with 50% (mass/volume, w/v) acetic acid. The reaction system was allowed to react at a constant temperature of 30℃for 24 hours.
After the reaction was completed, the target product in the reaction solution (same as in experimental example 1) was detected and analyzed by an HPLC method, and the detection method by HPLC was performed with reference to experimental example 1. As an example, fig. 3 and 4 show HPLC patterns of reaction solutions obtained by reacting a reaction system containing the pyruvate decarboxylase mutant M3 for 30min and 24h, respectively, and fig. 5 and 6 show HPLC patterns of reaction solutions obtained by reacting a reaction system containing the pyruvate decarboxylase mutant M15 for 30min and 24h, respectively.
The conversion data of the individual pyruvate decarboxylase mutants (M1 to M15) and the pyruvate decarboxylase of comparative example 1 on the substrate (benzaldehyde) are detailed in Table 4 below:
TABLE 4 regreen experimental data for pyruvate decarboxylase mutants M1 to M15 and pyruvate decarboxylase of comparative example 1
As can be seen from Table 4, in the re-screening experiments, the conversion of substrates by the pyruvate decarboxylase mutants M1 to M15 was significantly better than that of the pyruvate decarboxylase of comparative example 1. Taking the pyruvate decarboxylase mutant M15 as an example, the conversion rate of the pyruvate decarboxylase mutant M15 to the substrate can reach 95.2%, which is 38 times of the conversion rate of the pyruvate decarboxylase of the comparative example 1 to the substrate. In addition, for the pyruvate decarboxylase mutant obtained by adopting a single site mutation mode, the substrate conversion rate of the pyruvate decarboxylase mutant obtained by adopting a W551D point mutation mode is the highest, and then M475K is adopted; for the pyruvate decarboxylase mutant obtained by the multi-site combined mutation mode, the substrate conversion rate of the pyruvate decarboxylase mutant obtained by the T7R, L38M, W551D and M475K combined mutation mode is highest.
Experimental example 3 comparison of the conversion ratio of the recombinant engineering bacteria T1 to T15 to the substrate under the optimized Process conditions
Providing a clean three-neck flask with a magnetic stirring rotor, adding 55g of pure water and 6.4g of sodium pyruvate into the three-neck flask, placing the three-neck flask containing the pure water and the sodium pyruvate into a constant-temperature magnetic stirring water bath kettle at 30 ℃, starting stirring, and sequentially adding 100 mu L of Mg with the concentration of 2mol/L into the three-neck flask 2+ Buffer solution and 100. Mu.L of thiamine pyrophosphate (TPP) at a concentration of 50mg/mL to obtain a mixed system, adjusting the pH of the mixed system to 5.95.+ -. 0.2 with 10% (mass/volume, w/v) NaOH, then continuing to add 7.5mL of enzyme solution containing a single pyruvate decarboxylase mutant (prepared in example 3) or the pyruvate decarboxylase enzyme solution of comparative example 1 to the three-necked flask to obtain a pre-reaction system, and then slowly dropping 6g of benzaldehyde (dropping speed of 0.4 mL/h) into the pre-reaction system using a peristaltic pump to perform a catalytic reaction, wherein 50% (mass/volume, w/v) acetic acid was used to control the pH of the whole reaction system to 6.2.+ -. 0.2, and the reaction was carried out at a constant temperature of 30 ℃ for 24 hours.
After the reaction was completed, the target product in the reaction solution (same as in experimental example 1) was detected and analyzed by an HPLC method, and the detection method by HPLC was performed with reference to experimental example 1. As an example, fig. 7 and 8 show HPLC profiles of reaction solutions obtained by feeding benzaldehyde 1h and feeding benzaldehyde 24h to a pre-reaction system comprising the pyruvate decarboxylase mutant M3, respectively, and fig. 9 and 10 show HPLC profiles of reaction solutions obtained by feeding benzaldehyde 1h and feeding benzaldehyde 24h to a pre-reaction system comprising the pyruvate decarboxylase mutant M15, respectively.
The conversion data of the individual pyruvate decarboxylase mutants (M1 to M15) and the pyruvate decarboxylase of comparative example 1 on the substrate (benzaldehyde) are detailed in Table 5 below:
TABLE 5 optimization of pyruvate decarboxylase mutants M1 to M15 and pyruvate decarboxylase of comparative example 1 experimental data on process conditions
As can be seen from Table 5, in the experiments with optimized process conditions, the conversion of substrates by the pyruvate decarboxylase mutants M1 to M15 was significantly better than that of the pyruvate decarboxylase of comparative example 1. Taking pyruvic acid decarboxylase mutant M15 as an example, the conversion rate of the pyruvic acid decarboxylase mutant M15 to the substrate can reach 99.7%, which is 11 times of the conversion rate of the pyruvic acid decarboxylase of comparative example 1 to the substrate.
Experimental example 4 comparison of tolerance of pyruvate decarboxylase mutants M1 to M15 to substrate concentration
A clean three-necked flask equipped with a magnetic stirring rotor was provided, and 60g of pure water and 100. Mu.L of Mg having a concentration of 2mol/L were sequentially added to the three-necked flask 2+ Buffer solution and 100. Mu.L of thiamine pyrophosphate (TPP) at a concentration of 50mg/mL to obtain a mixed system, adjusting pH of the mixed system to 5.95.+ -. 0.2 with 10% (mass/volume, w/v) NaOH, then continuing to add 7.0mL of an enzyme solution containing a single pyruvate decarboxylase mutant (prepared in example 3) or a pyruvate decarboxylase enzyme solution of comparative example 1 to a three-necked flask, and adding a predetermined amount of benzaldehyde to obtain a pre-reaction system Four groups of pre-reaction systems are respectively arranged for each enzyme solution, and the four groups of pre-reaction systems are only different in that: the amounts of benzaldehyde added were different, the amount of benzaldehyde added in the first group of pre-reaction systems was 1g (corresponding to 1% of the concentration of benzaldehyde in the reaction system), the amount of benzaldehyde added in the second group of pre-reaction systems was 2g (corresponding to 2% of the concentration of benzaldehyde in the reaction system), the amount of benzaldehyde added in the third group of pre-reaction systems was 4g (corresponding to 3% of the concentration of benzaldehyde in the reaction system), and the amount of benzaldehyde added in the fourth group of pre-reaction systems was 6g (corresponding to 6% of the concentration of benzaldehyde in the reaction system). The three-neck flask containing the pre-reaction system is placed in a constant temperature magnetic stirring water bath kettle at 30 ℃, stirring is started for 1h, then 6.4g of sodium pyruvate is respectively added into each pre-reaction system to correspondingly obtain a reaction system, and 50% (mass/volume, w/v) acetic acid is adopted to adjust the pH value of the reaction system to 6.2+/-0.2. The reaction system was allowed to react at a constant temperature of 30℃for 2 hours.
After the reaction was completed, the target product in the reaction solution (same as in experimental example 1) was detected and analyzed by an HPLC method, and the detection method by HPLC was performed with reference to experimental example 1. The conversion data of the individual pyruvate decarboxylase mutants (M1 to M15) and the pyruvate decarboxylase of comparative example 1 for substrate (benzaldehyde) at different substrate (benzaldehyde) concentrations are shown in Table 6 below:
TABLE 6 tolerance test data for pyruvate decarboxylase mutants M1 to M15 and pyruvate decarboxylase of comparative example 1
As can be seen from table 6, the conversion rate of the pyruvate decarboxylase mutants M1 to M15 and the pyruvate decarboxylase of comparative example 1 to the substrate (benzaldehyde) all tended to decrease with increasing substrate concentration in the reaction system, but the conversion rate of the pyruvate decarboxylase mutants M1 to M15 to the substrate was significantly higher than that of the pyruvate decarboxylase of comparative example 1 at the same substrate concentration, indicating that: the substrate tolerance of the pyruvate decarboxylase mutants M1 to M15 was better than that of the pyruvate decarboxylase of comparative example 1.
When the substrate concentration in the reaction system was increased from 1% to 6%, the conversion rate of the corresponding pyruvate decarboxylase mutant M15 to the substrate was decreased by 69%, while the conversion rate of the pyruvate decarboxylase of comparative example 1 was decreased by 80%, indicating that the tolerance of the pyruvate decarboxylase mutant M15 to the substrate was significantly better than that of the pyruvate decarboxylase of comparative example 1, and when the substrate concentration in the reaction system was 6%, the conversion rate of the pyruvate decarboxylase mutant M15 to the substrate was 60 times that of the pyruvate decarboxylase of comparative example 1.
Experimental example 5 comparison of the thermostability of pyruvate decarboxylase mutants M1 to M15
Providing a clean three-neck flask with a magnetic stirring rotor, sequentially adding 55g of pure water and 6.4g of sodium pyruvate into the three-neck flask, placing the three-neck flask containing the pure water and the sodium pyruvate into a constant-temperature magnetic stirring water bath kettle at 30 ℃, starting stirring, and sequentially adding 6g of benzaldehyde and 100 mu L of Mg with the concentration of 2mol/L into the three-neck flask 2+ Buffer and 100. Mu.L of thiamine pyrophosphate (TPP) at a concentration of 50mg/mL to obtain a mixed system, pH of the mixed system was adjusted to 5.95.+ -. 0.2 with 10% (mass/volume, w/v) NaOH, and then 7.0mL of an enzyme solution containing a single pyruvate decarboxylase mutant (prepared in example 3) or a pyruvate decarboxylase enzyme solution of comparative example 1 was continuously added to a three-necked flask to obtain reaction systems, each enzyme solution was provided with two sets of reaction systems, respectively, differing in that the two sets of reaction systems were merely: one group of added enzyme solutions is not subjected to heat treatment, and the other group of added enzyme solutions is the enzyme solution after heat treatment at a constant temperature of 50 ℃ for 60 min. The pH of each reaction system was adjusted to 6.2.+ -. 0.2 with 50% (mass/volume, w/v) acetic acid, and the reaction system was allowed to react at a constant temperature of 30℃for 2 hours.
After the reaction was completed, the target product in the reaction solution (same as in experimental example 1) was detected and analyzed by an HPLC method, and the detection method by HPLC was performed with reference to experimental example 1. The conversion of the substrate (benzaldehyde) and the conversion of the substrate (benzaldehyde) after heat treatment of the respective pyruvate decarboxylase mutants (M1 to M15) and the pyruvate decarboxylase of comparative example 1 were described in Table 7 below:
TABLE 7 thermal stability test data for pyruvate decarboxylase mutants M1 to M15 and pyruvate decarboxylase of comparative example 1
As can be seen from Table 7, the thermostable performance of the pyruvate decarboxylase mutants M1 to M15 was significantly better than that of the pyruvate decarboxylase of comparative example 1. Although the substrate conversion rate after heat treatment was reduced as compared with that after heat treatment, the conversion rate of the substrate was reduced by 6.3% for the pyruvate decarboxylase mutants M1 to M15, taking the pyruvate decarboxylase mutant M15 as an example, whereas the pyruvate decarboxylase of comparative example 1 was substantially lost in catalytic activity for the substrate after heat treatment.
From experimental examples 1 to 5, the comprehensive properties of the pyruvate decarboxylase mutants M1 to M15 are obviously better than those of the pyruvate decarboxylase of comparative example 1, and the pyruvate decarboxylase mutants M1 to M15 have the advantages of higher catalytic activity, better optical selectivity and better stability than the pyruvate decarboxylase of comparative example 1.
The application provides a pyruvic acid decarboxylase mutant and application thereof in preparing alpha-hydroxyketone compounds. The principles and embodiments of the present application have been described herein with reference to specific examples, the description of the above examples is only for aiding in understanding the technical solution of the present application and its core ideas; those of ordinary skill in the art will appreciate that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the technical solutions according to the embodiments of the present application.
Sequence listing
<110> Taizhou enzyme Biotechnology Co., ltd
<120> pyruvate decarboxylase mutant and application thereof in preparing alpha-hydroxyketone compound
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Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
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Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
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Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
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Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
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Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
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Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
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Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
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Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
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50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Ile Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 4
<211> 1707
<212> DNA
<213> artificial sequence
<400> 4
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tctatcttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 5
<211> 568
<212> PRT
<213> artificial sequence
<400> 5
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Met Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 6
<211> 1707
<212> DNA
<213> artificial sequence
<400> 6
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatggaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 7
<211> 568
<212> PRT
<213> artificial sequence
<400> 7
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Asp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 8
<211> 1707
<212> DNA
<213> artificial sequence
<400> 8
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa gatggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 9
<211> 568
<212> PRT
<213> artificial sequence
<400> 9
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Lys Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 10
<211> 1707
<212> DNA
<213> artificial sequence
<400> 10
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccacta agtggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 11
<211> 568
<212> PRT
<213> artificial sequence
<400> 11
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Leu His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 12
<211> 1707
<212> DNA
<213> artificial sequence
<400> 12
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgctgca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 13
<211> 568
<212> PRT
<213> artificial sequence
<400> 13
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Pro Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 14
<211> 1707
<212> DNA
<213> artificial sequence
<400> 14
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gccctgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 15
<211> 568
<212> PRT
<213> artificial sequence
<400> 15
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Lys Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 16
<211> 1707
<212> DNA
<213> artificial sequence
<400> 16
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttaagatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 17
<211> 568
<212> PRT
<213> artificial sequence
<400> 17
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Lys Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Asp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 18
<211> 1707
<212> DNA
<213> artificial sequence
<400> 18
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttaagatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa gatggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 19
<211> 568
<212> PRT
<213> artificial sequence
<400> 19
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Ala Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Tyr Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 20
<211> 1707
<212> DNA
<213> artificial sequence
<400> 20
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tctgcattca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attgtataaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 21
<211> 568
<212> PRT
<213> artificial sequence
<400> 21
Met Ser Tyr Thr Val Gly Thr Trp Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Met Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 22
<211> 1707
<212> DNA
<213> artificial sequence
<400> 22
atgagttata ctgtcggtac ctggttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctatgaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 23
<211> 568
<212> PRT
<213> artificial sequence
<400> 23
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Tyr Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Glu Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 24
<211> 1707
<212> DNA
<213> artificial sequence
<400> 24
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgctatact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagaa gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 25
<211> 568
<212> PRT
<213> artificial sequence
<400> 25
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Met Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Lys Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Asp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 26
<211> 1707
<212> DNA
<213> artificial sequence
<400> 26
atgagttata ctgtcggtac ctatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tatgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttaagatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa gatggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 27
<211> 568
<212> PRT
<213> artificial sequence
<400> 27
Met Ser Tyr Thr Val Gly Arg Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Met Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gly Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 28
<211> 1707
<212> DNA
<213> artificial sequence
<400> 28
atgagttata ctgtcggtcg atatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tatgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctggtatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttatgatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 29
<211> 568
<212> PRT
<213> artificial sequence
<400> 29
Met Ser Tyr Thr Val Gly Arg Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Lys Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Tyr Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 30
<211> 1707
<212> DNA
<213> artificial sequence
<400> 30
atgagttata ctgtcggtcg atatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tttgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttaagatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa tggggttatc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 31
<211> 568
<212> PRT
<213> artificial sequence
<400> 31
Met Ser Tyr Thr Val Gly Arg Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Met Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Thr Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Asp Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Thr Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Ala Asn
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ser Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Lys Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Ala Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Asp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 32
<211> 1707
<212> DNA
<213> artificial sequence
<400> 32
atgagttata ctgtcggtcg atatttagcg gagcggcttg tccagattgg tctcaagcat 60
cacttcgcag tcgcgggcga ctacaacctc gtccttcttg acaacctgct tatgaacaaa 120
aacatggagc aggtttattg ctgtaacgaa ctgaactgcg gtttcagtgc agaaggttat 180
gctcgtgcca aaggcgcagc agcagccgtc gttacctaca gcgttggtgc gctttccgca 240
tttgatgcta tcggtggcgc ctatgcagaa aaccttccgg ttatcctgat ctccggtgct 300
ccgaacaaca acgaccacgc tgctggtcat gtgttgcatc atgctcttgg caaaaccgac 360
tatcactatc agttggaaat ggccaagaac atcacggccg ccgctgaagc gatttacacc 420
ccggaagaag ctccggctaa aatcgatcac gtgatcaaaa ctgctcttcg cgagaagaag 480
ccggtttatc tcgaaatcgc ttgcaacact gcttccatgc cctgcgccgc tcctggaccg 540
gcaagtgcat tgttcaatga cgaagccagc gacgaagcat ccttgaatgc agcggttgac 600
gaaaccctga aattcatcgc caaccgcgac aaagttgccg tcctcgtcgg cagcaagctg 660
cgcgctgctg gtgctgaaga agctgctgtt aaattcaccg acgctttggg cggtgcagtg 720
gctactatgg ctgctgccaa gagcttcttc ccagaagaaa atgccaatta cattggtacc 780
tcatggggcg aagtcagcta tccgggcgtt gaaaagacga tgaaagaagc cgatgcggtt 840
atcgctctgg ctcctgtctt caacgactac tccaccactg gttggacgga tatccctgat 900
cctaagaaac tggttctcgc tgaaccgcgt tctgtcgttg tcaacggcat tcgcttcccc 960
agcgttcatc tgaaagacta tctgacccgt ttggctcaga aagtttccaa gaaaaccggt 1020
tctttggact tcttcaaatc cctcaatgca ggtgaactga agaaagccgc tccggctgat 1080
ccgagtgctc cgttggtcaa cgcagaaatc gcccgtcagg tcgaagctct tctgaccccg 1140
aacacgacgg ttattgctga aaccggtgac tcttggttca atgctcagcg catgaagctc 1200
ccgaacggtg ctcgcgttga atatgaaatg cagtggggtc acattggttg gtccgttcct 1260
gccgccttcg gttatgccgt cggtgctccg gaacgtcgca acatcctcat ggttggtgat 1320
ggttccttcc agctgacggc tcaggaagtt gctcagatgg ttcgcctgaa actgccggtt 1380
atcatcttct tgatcaataa ctatggttac accatcgaag ttaagatcca tgatggtccg 1440
tacaacaaca tcaagaactg ggattatgcc ggtctgatgg aagtgttcaa cggtaacggt 1500
ggttatgaca gcggtgctgc taaaggcctg aaggctaaaa ccggtggcga actggcagaa 1560
gctatcaagg ttgctctggc aaacaccgac ggcccaaccc tgatcgaatg cttcatcggt 1620
cgtgaagact gcactgaaga attggtcaaa gatggtaagc gcgttgctgc cgccaacagc 1680
cgtaagcctg ttaacaagct cctctag 1707
<210> 33
<211> 30
<212> DNA
<213> artificial sequence
<400> 33
attttcaatg ctcagcgcat gaagctcccg 30
<210> 34
<211> 30
<212> DNA
<213> artificial sequence
<400> 34
agagtcaccg gtttcagcaa taaccgtcgt 30

Claims (12)

1. A pyruvate decarboxylase mutant, characterized in that the amino acid sequence of the pyruvate decarboxylase mutant is identical to SEQ ID NO:1, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similarity, and the amino acid sequence of the pyruvate decarboxylase mutant is represented by SEQ ID NO:1, wherein the amino acid sequence is obtained by one or more point mutations in the amino acid sequence shown in 1; the pyruvate decarboxylase mutant has pyruvate decarboxylase activity.
2. The pyruvate decarboxylase mutant according to claim 1, characterized by the fact that the amino acid at which the point mutation takes place comprises the amino acid sequence of SEQ ID NO:1, at least one of amino acid T at position 7, amino acid T at position 8, amino acid L at position 38, amino acid N at position 169, amino acid a at position 246, amino acid G at position 294, amino acid W at position 392, amino acid V at position 450, amino acid Q at position 452, amino acid I at position 472, amino acid M at position 475, amino acid I at position 476, amino acid V at position 549, amino acid W at position 551, amino acid K at position 553, or amino acid V at position 555.
3. The pyruvate decarboxylase mutant of claim 2 wherein the point mutation is effected by at least one of T7R, T8W, L M, N169Y, A246M, G294K, W A, V450E, Q452G, I472M, M475K, I476L, V549Y, W551D, K553Y or V555P.
4. A pyruvate decarboxylase mutant according to claim 3 characterized by the fact that the point mutation occurs in any of the following ways:
(1)W392I；
(2)I472M；
(3)W551D；
(4)G294K；
(5)I476L；
(6)V555P；
(7)M475K；
(8) W551D and M475K;
(9) V549Y and W392A;
(10) T8W and a246M;
(11) N169Y and V450E;
(12) M475K, L M and W551D;
(13) T7R, L M and Q452G;
(14) T7R, K Y and M475K; or (b)
(15) T7R, L M, W551D and M475K.
5. A pyruvate decarboxylase mutant according to claim 3 characterized by its amino acid sequence selected from the group consisting of SEQ ID NOs: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13. SEQ ID NO: 15. SEQ ID NO: 17. SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO: 23. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO:29 or SEQ ID NO:31, and a polypeptide comprising the amino acid sequence shown in any one of seq id no.
6. A nucleic acid molecule comprising a nucleotide sequence encoding a pyruvate decarboxylase mutant according to any one of claims 1 to 5.
7. The nucleic acid molecule of claim 6, wherein said nucleotide sequence is selected from the group consisting of SEQ ID NOs: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 14. SEQ ID NO: 16. SEQ ID NO: 18. SEQ ID NO: 20. SEQ ID NO: 22. SEQ ID NO: 24. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO:30 or SEQ ID NO:32, or a nucleotide sequence as set forth in any one of seq id no.
8. A recombinant expression vector comprising a vector and the nucleic acid molecule of claim 6 or 7; the vector is selected from a plasmid, cosmid, phage or viral vector.
9. A recombinant expression transformant comprising a host, and the nucleic acid molecule according to claim 6 or 7, or the recombinant expression vector according to claim 8, introduced into the host; the host is selected from eukaryotes or prokaryotes.
10. A method for preparing a pyruvate decarboxylase mutant, characterized by culturing the recombinant expression transformant of claim 9, and obtaining said pyruvate decarboxylase mutant from the culture.
11. A method for preparing chiral alpha-hydroxy ketone compound, characterized in that the pyruvate decarboxylase mutant as defined in any one of claims 1 to 5 or the pyruvate decarboxylase mutant prepared by the preparation method as defined in claim 10 is used as a catalyst, and the first compound and the second compound are contacted and reacted to generate alpha-hydroxy ketone compound;
wherein the first compound has a structure represented by the following general formula (I):
in the general formula (I), R 1 Selected from hydrogen atoms or hydroxyl groups, and X is selected from hydrogen atoms or monovalent metal ions.
The second compound has a structure represented by the following general formula (II):
in the general formula (II), R 2 Selected from aryl or heteroaryl groups.
12. The method according to claim 11, wherein the first compound is at least one selected from pyruvic acid, hydroxypyruvic acid and sodium pyruvate, and the second compound is benzaldehyde.
CN202210361627.4A 2022-04-07 2022-04-07 Pyruvic acid decarboxylase mutant and application thereof in preparation of alpha-hydroxyketone compound Active CN116790569B (en)

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