CN113584105B - Method for preparing puromycin by enzyme method - Google Patents

Abstract

The invention relates to the technical field of medicine synthesis, in particular to a method for preparing puromycin by an enzyme method. The method comprises the following steps: under the action of methyl thioribose kinase and ATP, the 3-amino-D-ribose generates beta-aminoribose-1-phosphate; beta-aminoribose-1-phosphate and N, N-dimethyl purine generate 3-amino-N, N-dimethyl adenosine under the action of purine nucleoside phosphorylase; 3-amino-N, N-dimethyl adenosine and methyl tyrosine produce puromycin under the action of amino acid ligase and ATP. Aiming at the limitations of puromycin fermentation and chemical preparation methods, the invention develops an enzymatic preparation process of puromycin. Compared with the traditional route, the process has the advantages of short route, high conversion rate, mild reaction conditions, environmental protection and the like, which not only remarkably reduces the production cost of puromycin, but also improves the safety and green index in industrial production.

Description

Method for preparing puromycin by enzyme method

Technical Field

The invention relates to the technical field of medicine synthesis, in particular to a method for preparing puromycin by an enzyme method.

Background

Puromycin (Puromycin) is an aminoglycoside antibiotic produced by the fermentative metabolism of streptomyces albidojensis (Streptomyces alboniger) and kills gram positive bacteria, various animal and insect cells by inhibiting protein synthesis. Coli in a particular situation; are commonly used to screen eukaryotic or prokaryotic polyclonal or monoclonal cells capable of expressing the pac gene (puror) by plasmid transfection/transformation, viral infection, and the like. Puromycin is used not only for the screening of stable cell lines but also for the maintenance of stable cell lines. Puromycin is characterized by rapid cell action, typically within 2 days, killing 99% of cells that do not express the pac gene. In gram positive bacteria, animal or insect cells puromycin inhibits or kills cells by inhibiting protein synthesis. The mechanism of action is that puromycin is an analogue of the 3' end of the aminoacyl-tRNA molecule, capable of binding to the A-site of the ribosome and incorporating into an extended peptide chain. Puromycin, after binding to the A-site, does not participate in any subsequent reaction, leading to premature termination of protein synthesis and release of the C-terminus.

Traditional methods for preparing puromycin include fermentation methods and chemical synthesis methods:

1) Fermentation method: streptomyces nigrogenicus is used for fermentation production, but the yield is too low, and the purification process is complex due to a large number of byproducts, so the production cost is high.

2) Chemical synthesis method: morris J.Robin reports the chemical synthesis of puromycin using 3-azido-adenosine as a starting material and 6 steps of chemical catalysis to produce puromycin with a total yield of about 26%. The chemical synthesis method of puromycin needs to protect and deprotect a plurality of functional groups, the whole route is long, and various toxic and harmful reagents (such as TMSCl, pyridine, DCC and the like) are needed in the chemical preparation process, so that the safety coefficient in the production process is low, the environmental compatibility is low, the whole yield is not high, and the final production cost is high.

With public emphasis on personal safety and natural environment protection in industrial production, the green chemical industry is a necessary trend of development. As a part of biocatalysis, enzyme catalysis is compatible with the characteristics of high chemical catalytic concentration, environment protection of fermentation production, mild conditions and the like, and is becoming an important direction of green chemical development. The enzymatic method for preparing puromycin is reported.

Disclosure of Invention

In view of this, the present invention provides a method for preparing puromycin by an enzymatic method. The method can realize the efficient conversion from the aminoribose to the puromycin by utilizing three-step enzyme reaction, so that the method has outstanding advantages in various aspects such as production cost, environmental protection, waste gas and waste water emission and the like.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for preparing puromycin by an enzymatic method, which comprises the following steps:

under the action of methyl thioribose kinase and ATP, the 3-amino-D-ribose generates beta-aminoribose-1-phosphate;

beta-aminoribose-1-phosphate and N, N-dimethyl purine generate 3-amino-N, N-dimethyl adenosine under the action of purine nucleoside phosphorylase;

3-amino-N, N-dimethyl adenosine and methyl tyrosine produce puromycin under the action of amino acid ligase and ATP.

The invention utilizes methyl thioribokinase (Kinase, EC 2.7.1.100) to phosphorylate 3-amino-D-ribose to obtain corresponding beta-aminoribosyl-1-phosphate, then purine nucleoside phosphorylase (PNP, EC 2.4.2.1) is used for converting the corresponding beta-aminoribosyl-1-phosphate into 3-amino-N, N-dimethyl adenosine under the action of N, N-dimethyl purine, and finally (aaLigase, EC 6.3.2.28) is used for condensing methylated tyrosine under the action of amino acid ligase to obtain the final puromycin.

In the present invention, the purine nucleoside phosphorylase is PNP-a and/or PNP-b.

In the present invention, the amino acid ligase is aaligase-1 and/or aaligase-2.

Preferably, the purine nucleoside phosphorylase is PNP-b and the amino acid ligase is aaligase-1.

Preferably, the reaction system in which ATP participates further includes an ATP circulating system composed of acetate kinase and acetyl phosphate.

In the first and third steps, adenosine Triphosphate (ATP) is needed, so that an ATP circulating system consisting of acetate kinase (AK, EC 2.7.2.1)/acetyl phosphate (AcP) is added into the whole system, thereby greatly reducing the consumption of expensive adenosine triphosphate and further reducing the production cost.

Preferably, the preparation method of the invention is a multi-component one-pot method.

Preferably, the ratio of 3-amino-D-ribose, N-dimethylpurine, methyltyrosine, ATP, methylthioribose kinase, purine nucleoside phosphorylase, amino acid ligase in the multicomponent one-pot reaction system is (50-150) mM: (50-150) mM: (50-150) mM: (1-2) mM: (1000-1500) U: (800-1500) U: (800-1500) U;

alternatively, the ratio of 3-amino-D-ribose, N-dimethylpurine, methyltyrosine, ATP, acetyl phosphate, methylthioribose kinase, purine nucleoside phosphorylase, amino acid ligase, acetate kinase is (50-150) mM: (50-150) mM: (50-150) mM: (1-2) mM: (100-300) mM: (1000-1500) U: (800-1500) U: (800-1500) U: (2000-4000) U.

Preferably, in the multicomponent one-pot reaction system, the ratio of 3-amino-D-ribose, N-dimethylpurine, methyltyrosine, ATP, methylthioribose kinase, purine nucleoside phosphorylase and amino acid ligase is (50-150) mM: (50-150) mM: (50-150) mM:1.5mM: (1000-1500) U: (800-1500) U: (800-1500) U;

alternatively, the ratio of 3-amino-D-ribose, N-dimethylpurine, methyltyrosine, ATP, acetyl phosphate, methylthioribose kinase, purine nucleoside phosphorylase, amino acid ligase, acetate kinase is (50-150) mM: (50-150) mM: (50-150) mM:1.5mM: (100-300) mM: (1000-1500) U: (800-1500) U: (800-1500) U: (2000-4000) U.

Preferably, the multi-component one-pot reaction system also comprises an activator and a substrate cosolvent;

preferably, the activator is magnesium chloride and the substrate cosolvent is isopropanol.

Preferably, the ratio of 3-amino-D-ribose, N-dimethylpurine, methyl tyrosine, ATP, activator, substrate cosolvent, methyl thioribose kinase, purine nucleoside phosphorylase and amino acid ligase in the multicomponent one-pot reaction system is (50-150) mM: (50-150) mM: (50-150) mM: (1-2) mM: (5-15) mM: (100-150) mL: (1000-1500) U: (800-1500) U: (800-1500) U;

alternatively, the ratio of 3-amino-D-ribose, N-dimethylpurine, methyltyrosine, ATP, acetyl phosphate, activator, substrate cosolvent, methyl thioribose kinase, purine nucleoside phosphorylase, amino acid ligase, acetate kinase is (50-150) mM: (50-150) mM: (50-150) mM: (1-2) mM: (100-300) mM: (5-15) mM: (100-150) mL: (1000-1500) U: (800-1500) U: (800-1500) U: (2000-4000) U.

Preferably, in the multicomponent one-pot reaction system, the ratio of 3-amino-D-ribose, N-dimethylpurine, methyl tyrosine, ATP, activator, substrate cosolvent, methyl thioribose kinase, purine nucleoside phosphorylase and amino acid ligase is (50-150) mM: (50-150) mM: (50-150) mM:1.5mM:10mM: (100-150) mL: (1000-1500) U: (800-1500) U: (800-1500) U;

alternatively, the ratio of 3-amino-D-ribose, N-dimethylpurine, methyltyrosine, ATP, acetyl phosphate, activator, substrate cosolvent, methyl thioribose kinase, purine nucleoside phosphorylase, amino acid ligase, acetate kinase is (50-150) mM: (50-150) mM: (50-150) mM:1.5mM: (100-300) mM:10mM: (100-150) mL: (1000-1500) U: (800-1500) U: (800-1500) U: (2000-4000) U.

Preferably, the reaction conditions of the multicomponent one-pot process are: the pH value is 7.0-8.5, and the mixture is stirred for 2-10 hours at 15-30 ℃.

In the specific examples provided herein, the reaction conditions for the multicomponent one-pot process are: the pH value is 7.0-8.5, and the mixture is stirred for 4 hours at room temperature.

In the invention, the method further comprises the steps of enzyme removal, separation, purification and crystallization by using a nonpolar column Seplite D101 after the reaction is finished.

Preferably, the crystallization is crystallization in an aqueous ethanol solution.

In a specific embodiment provided herein, the crystallization is in ethanol/water (3:2, v:v).

The invention provides a method for preparing puromycin by an enzyme method. The method comprises the following steps: under the action of methyl thioribose kinase and ATP, the 3-amino-D-ribose generates beta-aminoribose-1-phosphate; beta-aminoribose-1-phosphate and N, N-dimethyl purine generate 3-amino-N, N-dimethyl adenosine under the action of purine nucleoside phosphorylase; 3-amino-N, N-dimethyl adenosine and methyl tyrosine produce puromycin under the action of amino acid ligase and ATP. The invention has the following technical effects:

aiming at the limitations of puromycin fermentation and chemical preparation methods, the invention develops an enzymatic preparation process of puromycin. Compared with the traditional route, the process has the advantages of short route, high conversion rate, mild reaction conditions, environmental protection and the like, which not only remarkably reduces the production cost of puromycin, but also improves the safety and green index in industrial production.

Drawings

FIG. 1 shows the route of the multi-step enzymatic method of the present invention for puromycin;

as shown in FIG. 1, the route uses 3-amino-D-ribose as a raw material, and is phosphorylated under the action of kinase (kinase) to obtain aminoribose-1-phosphate, then 3-amino-N, N-dimethyl adenosine analogue is generated under the action of Purine Nucleoside Phosphorylase (PNP), and finally puromycin is generated under the action of amino acid ligase (aaLigase).

Detailed Description

The invention discloses a method for preparing puromycin by an enzyme method, and a person skilled in the art can properly improve the technological parameters by referring to the content of the puromycin. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.

Enzyme related information:

methyl thioribose Kinase (Kinase): from Bacillus subtilis (Bacillus subtilis) (Uniprot ID: O31663, EC 2.7.1.100);

purine nucleoside phosphorylase PNP (PNP-a & PNP-b) is modified by a purine nucleoside phosphorylase 2 in Escherichia coli (Uniprot ID: P45563, EC 2.4.2.1);

amino acid ligase aaLigase (aaLigase-1 & aaLigase-2): is mutated by a L-amino acid ligase with a very broad catalytic substrate in Pseudomonas syringae (UniprotID: Q842E2, EC 6.3.2.28);

acetate kinase AK: is derived from Thermotoga maritima (Thermotoga maritima) (Uniprot ID: Q9WYB1, EC 2.7.2.1).

The amino acid sequence and DNA sequence of the enzyme are as follows:

fermentation production of enzyme:

the enzyme required by the patent is prepared by constructing a specific expression plasmid after the company synthesizes the corresponding genes and then fermenting and producing the specific expression plasmid by escherichia coli. The method specifically comprises the following steps:

after sequence optimization, the genes corresponding to the enzymes are synthesized by general biological company (Chuzhou Anhui, anhui), ndeI/XhoI restriction sites are introduced and subcloned into pET 28a expression vectors. Plasmid with correct sequence was confirmed to be transferred into E.coli (BL 21) competent cells plate culture (of the species Prinsepia) and monoclonal miniculture, the bacteria with correct protein expression are finally amplified and cultured step by step. Specifically, the single colony is transferred into 5mL LB culture solution (37 ℃) containing 50 mu M kanamycin for culture, and when the cell grows to the logarithmic phase, the cell is inoculated into 250mL LB culture solution containing the same antibiotics, and when the cell grows to the logarithmic phase, the cell is transferred into a 5L culture fermentation tank for culture, and the final protein expression is carried out. In 5L fermenter culture, 0.5mM isopropyl-. Beta. -D-thiogalactopyranoside (IPTG) was added at 25℃at cell OD of about 20 to induce protein expression for 6 hours, and finally the cells were collected by high-speed centrifugation (4000 rpm,20 min) to obtain 30-65g of wet cells over-expressing the enzyme. A small amount of cells are firstly mixed with a buffer solution (50 mM, pH 8.0) of tris (hydroxymethyl) aminomethane hydrochloride (Tris.HCl) on an ice basin uniformly, then the cells are broken by a freeze thawing method, and clear liquid is subjected to SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) after cell walls are removed by high-speed centrifugation to determine protein expression. Cells with correct protein expression were used for the next catalytic experiment. Specifically, the remaining cells were mixed with Tris.HCl buffer (50 mM, pH 8.0) at a low temperature uniformly (about 10 g of wet cells: 200mL of buffer), followed by crushing the cell walls at a low temperature of Gao Yapo and removing the cell walls by high-speed centrifugation (16000 rpm,45 min) to obtain an enzyme-containing supernatant for use (the enzyme activity obtained was 100 to 220U/mL, U was the amount of enzyme required for converting 1. Mu. Mol of substrate at room temperature for one minute). LB medium consisted of: 1% tryptone, 0.5% yeast powder, 1% NaCl,1% dipotassium hydrogen phosphate and 5% glycerol.

The raw materials or reagents used in the present invention are all commercially available.

The invention is further illustrated by the following examples:

example 1: preparation of aqueous solutions of Acetylphosphoric acid

135mL of phosphoric acid (85%, 2.0 mol) was dissolved in 1.2L of ethyl acetate, and then cooled to 0deg.C; to this solution was slowly added dropwise 376mL of cooled acetic anhydride (4.0 mol). The mixture was stirred at 0deg.C for 6 hours and poured into a 5 liter reaction flask containing 1 liter of water, 500 grams of ice and 168 grams of sodium bicarbonate. The mixture was stirred at low temperature until no bubbles were generated. The upper ethyl acetate phase was separated and discarded, and the remaining aqueous phase was adjusted to a pH of about 3 and extracted twice with 2.0L of ethyl acetate and 1.0L of ethyl acetate to remove most of the residual acetic acid. Finally, sodium hydroxide is used for adjusting the pH value of the aqueous solution containing the acetyl phosphate to be neutral for standby, and 1.5L of 1.2N of the aqueous solution of the acetyl phosphate is obtained through enzyme activity test.

Example 2: one-pot method for preparing puromycin by liquid enzyme (Kinase, PNP-a, aaligase-1, AK)

To 1L 100mM Tris-HCl (pH 8.0) solution was added 14.9 g 3-amino-D-ribose (100 mM), 16.2 g N, N-dimethylpurine nicotinic acid (100 mM), 19.5 g methyl tyrosine, 0.95 g MgCl ₂ (10 mM), 0.79 g of adenosine triphosphateMono sodium salt (1.5 mM), 170mL of the 1.2N aqueous acetyl phosphate solution (200 mM) prepared above, and 100mL of isopropanol (substrate cosolvent). Before enzyme is added, the pH of the solution is adjusted to 8.0, then 1500U of kinase, 1500UPNP-a, 800U of aaligase-1 and 3000U of AK are added to start enzyme reaction, the pH of the reaction solution is maintained between 7.0 and 8.5 in the reaction process, the reaction solution is stirred for 4 hours at room temperature, the pH of the solution is acidified to 3.0 to denature and precipitate the enzyme in the reaction solution, the pH value is adjusted to 7.0, then the reaction solution is put on a nonpolar column Seplite D101 (Silan materials and technologies Co., ltd.) for separation and purification, and the crude product puromycin is further crystallized in ethanol/water (3:2, v:v) to obtain 29.2 g of light yellow solid (the total yield is 62%).

Example 3: one-pot method for preparing puromycin by liquid enzyme (Kinase, PNP-b, aaligase-2, AK)

Example 3 uses PNP-b and aaligase-2, but not PNP-a and aaligase-1, similar to example 2.

To 1L 100mM Tris-HCl (pH 8.0) solution was added 7.45 g 3-amino-D-ribose (50 mM), 8.1 g N, N-dimethylpurine nicotinic acid (50 mM), 9.75 g methyl tyrosine, 0.95 g MgCl ₂ (10 mM), 0.79 g of adenosine triphosphate monosodium salt (1.5 mM), 85mL of the 1.2N aqueous acetyl phosphate solution prepared above (100 mM) and 100mL of isopropanol (substrate co-solvent). Before enzyme is added, the pH of the solution is adjusted to 8.0, then 1000U of kinase, 800U of PNP-b, 1500U of aaligase-2 and 2000U of AK are added to start enzyme reaction, the pH of the reaction solution is maintained between 7.0 and 8.5 in the reaction process, the reaction solution is stirred at room temperature for 6 hours, the pH of the solution is acidified to 3.0 to denature and precipitate the enzyme in the reaction solution, the pH value is adjusted back to 7.0, then the reaction solution is separated and purified by a nonpolar column Seplite D101 (SiAN blue Xiao technology New material Co., ltd.), and the crude product puromycin is further crystallized in ethanol/water to obtain 15.4 g of light yellow solid (total yield 65%).

Example 4: one-pot method for preparing puromycin by liquid enzyme (Kinase, PNP-b, aaligase-1, AK)

Similar to examples 2 and 3 above, PNP-b, aaligase-1, which had the best catalytic effect, was used for subsequent catalysis.

In 1L 100mM Tris-HCl solution (pH 8.0)After addition of 22.4 g of 3-amino-D-ribose (150 mM), 24.3 g of N, N-dimethylpurine nicotinic acid (150 mM), 29.3 g of methyl tyrosine, 0.95 g of MgCl ₂ (10 mM), 0.79 g of adenosine triphosphate monosodium salt (1.5 mM), 255mL of the 1.2N aqueous acetyl phosphate solution prepared above (300 mM) and 150mL of isopropanol (substrate co-solvent). Before enzyme is added, the pH of the solution is adjusted to 8.0, then 1200U of kinase, 1200U of PNP-b, 1200U of aaligase-1 and 4000U of AK are added to start enzyme reaction, the pH of the reaction solution is maintained between 7.0 and 8.5 in the reaction process, the reaction solution is stirred at room temperature for 6 hours, the pH of the solution is acidified to 3.0 to denature and precipitate the enzyme in the reaction solution, the pH value is adjusted back to 7.0, then the reaction solution is separated and purified by a nonpolar column Seplite D101 (SiAN blue Xiao technology New material Co., ltd.), and 60 g of pale yellow solid (the total yield is 82%) is obtained by further crystallizing the product puromycin crude product in ethanol/water.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

<110> Shenzhen Ruidelin Biotechnology Co., ltd

<120> method for preparing puromycin by enzymatic method

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Ala Val Ala Leu Ala Val Lys Leu Gly Leu Phe Pro Ser Lys Ser Thr

20 25 30

Leu Thr Cys Gln Glu Ile Gly Asp Gly Asn Leu Asn Tyr Val Phe His

35 40 45

Ile Tyr Asp Gln Glu His Asp Arg Ala Leu Ile Ile Lys Gln Ala Val

50 55 60

Pro Tyr Ala Lys Val Val Trp Glu Ser Trp Pro Leu Thr Ile Asp Arg

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Ala Arg Ile Glu Ser Ser Ala Leu Ile Arg Gln Gly Glu His Val Pro

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His Leu Val Pro Arg Val Phe Tyr Ser Asp Thr Glu Met Ala Val Thr

100 105 110

Val Met Glu Asp Leu Ser His Leu Lys Ile Ala Arg Lys Gly Leu Ile

115 120 125

Glu Gly Tyr Asn Tyr Pro His Leu Ser Gln His Ile Gly Glu Phe Leu

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Gly Lys Thr Leu Phe Tyr Ser Ser Asp Tyr Ala Leu Glu Pro Lys Val

145 150 155 160

Lys Lys Gln Leu Val Lys Gln Phe Thr Asn Pro Glu Leu Cys Asp Ile

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Thr Glu Arg Leu Val Phe Thr Asp Pro Phe Phe Asp His Asp Thr Asn

180 185 190

Asp Phe Glu Glu Glu Leu Arg Pro Phe Val Glu Lys Leu Trp Asn Asn

195 200 205

Asp Ser Val Lys Ile Glu Ala Ala Lys Leu Lys Lys Ser Phe Leu Thr

210 215 220

Ser Ala Glu Thr Ser Ile His Gly Asp Leu His Thr Gly Ser Ile Phe

225 230 235 240

Ala Ser Glu His Glu Thr Lys Val Met Asp Pro Glu Phe Ala Phe Tyr

245 250 255

Gly Pro Ile Gly Phe Asp Val Gly Gln Phe Ile Ala Asn Leu Phe Leu

260 265 270

Asn Ala Leu Ser Arg Asp Gly Ala Asp Arg Glu Pro Leu Tyr Glu His

275 280 285

Val Asn Gln Val Trp Glu Thr Phe Glu Glu Thr Phe Ser Glu Ala Trp

290 295 300

Gln Lys Asp Ser Leu Asp Val Tyr Ala Asn Ile Asp Gly Tyr Leu Thr

305 310 315 320

Asp Thr Leu Ser His Ile Phe Glu Glu Ala Ile Gly Phe Ala Gly Cys

325 330 335

Glu Leu Ile Arg Arg Thr Ile Gly Leu Ala His Val Ala Asp Leu Asp

340 345 350

Thr Ile Val Pro Phe Asp Lys Arg Ile Gly Arg Lys Arg Leu Ala Leu

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Glu Thr Gly Thr Ala Phe Ile Glu Lys Arg Ser Glu Phe Lys Thr Ile

370 375 380

Thr Asp Val Ile Glu Leu Phe Lys Leu Leu Val Lys Glu

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Met Ser Gln Val Gln Phe Ser His Asn Pro Leu Phe Cys Ile Trp Lys

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Ile Lys Thr Tyr Lys Pro Asp Phe Thr Val Arg Val Ala Phe Ile Leu

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Gly Ser Gly Leu Phe Ala Leu Ala Asp Gln Ile Glu Asn Ala Val Ala

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Ile Ser Tyr Glu Lys Leu Pro Gly Phe Pro Val Ser Thr Val His Gly

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Ala Ala Gly Glu Leu Glu Val Gly His Leu Gln Gly Val Pro Val Val

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Cys Met Lys Gly Arg Gly His Phe Tyr Thr Gly Arg Gly Met Thr Ile

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Met Thr Asp Ala Ile Arg Asn Phe Lys Leu Leu Gly Cys Glu Leu Leu

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Phe Cys Thr Asn Ala Ala Gly Ser Leu Arg Pro Glu Arg Gly Ala Gly

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Ser Leu Val Ala Leu Lys Asp His Ile Asn Thr Met Leu Met Thr Pro

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Met Val Gly Leu Asn Asp Asp Arg Phe Val Glu Arg Phe Phe Ser Leu

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Ala Asn Ala Glu Asp Ala Glu Tyr Ile Ala Leu Leu Gln Lys Val Ala

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Lys Glu Glu Gly Phe Pro Leu Thr Pro Ser Val Phe Val Ser Tyr Pro

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Gly Pro Asn Phe Glu Thr Ala Ala Glu Ile Arg Met Met Gln Ile Ile

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Gly Gly Asp Val Val Gly Met Ser Val Val Pro Glu Val Ile Ser Ala

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Arg His Cys Asp Leu Lys Val Val Ala Val Ser Ala Ile Thr Asn Met

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Ala Ala Glu Leu Ser Lys Gln Asn Phe Ile Asn Leu Ile Cys Gly Phe

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Leu Arg Lys Ile Ala

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Met Ser Gln Val Gln Phe Ser His Asn Pro Leu Phe Cys Ile Trp Lys

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Ile Lys Thr Tyr Lys Pro Asp Phe Thr Gln Arg Val Ala Phe Ile Leu

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Gly Ser Gly Leu Phe Ala Leu Ala Asp Gln Ile Glu Asn Ala Val Ala

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Ile Ser Tyr Glu Lys Leu Pro Gly Phe Pro Val Ser Thr Val His Gly

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Ala Ala Gly Glu Leu Glu Val Gly His Leu Gln Gly Val Pro Val Val

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Cys Met Lys Gly Arg Cys Gly Pro Tyr Glu Gly Arg Gly Met Thr Ile

85 90 95

Met Thr Asp Ala Ile Arg Thr Phe Lys Leu Leu Gly Cys Glu Leu Leu

100 105 110

Phe Cys Thr Asn Ala Ala Gly Ser Leu Arg Pro Glu Val Gly Ala Phe

115 120 125

Ser Leu Val Ala Leu Lys Asp His Ile Asn Thr Met Leu Met Thr Pro

130 135 140

Met Val Gly Leu Asn Asp Ile Arg Phe Gly Glu Arg Phe Phe Ser Leu

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Ala Asn Ala Glu Asp Ala Glu Tyr Arg Ala Asp Leu Gln Lys Val Ala

165 170 175

Lys Glu Glu Gly Phe Pro Leu Thr Pro Ser Val Phe Val Ser Tyr Pro

180 185 190

Gly Pro Asn Phe Glu Thr Ala Ala Glu Ile Arg Met Met Gln Ile Ile

195 200 205

Gly Gly Asp Val Val Gly Met Ser Val Val Pro Glu Val Ile Ser Ala

210 215 220

Arg His Cys Asp Leu Lys Val Val Ala Val Ser Ala Ile Thr Asn Met

225 230 235 240

Ala Glu Gly Leu Ser Asp Val Lys Leu Ser His Ala Gln Thr Leu Ala

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Ala Ala Glu Leu Ser Lys Gln Asn Phe Ile Asn Leu Ile Cys Gly Phe

260 265 270

Leu Arg Lys Ile Ala

275

<210> 4

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Met Thr Gln Ala Lys Glu Asn Ile Leu Val Tyr Val Asp Gly Tyr Ser

1 5 10 15

Ser Gly Ser Gln Leu Pro Thr Leu Val Ala Leu Ser Gly Trp Lys Cys

20 25 30

Val His Val Ser Ser Ser Ala Asn Pro Pro Glu Tyr Tyr Leu Arg Thr

35 40 45

Tyr His Lys Asp Glu Tyr Ile Ala His Phe Glu Tyr Gln Gly Asp Ile

50 55 60

Gln Ser Leu Ala Ser Ala Val Glu Ala Trp His Pro Ala Ala Val Leu

65 70 75 80

Arg Gly Ser Glu Ser Gly Val Ile Val Ala Asp Leu Leu Ala Ala Ala

85 90 95

Leu Asp Leu Gly Gly Asn Asp Pro Ser Thr Ser Leu Ala Arg Arg Asp

100 105 110

Lys Tyr Thr Met His Glu Ser Leu Lys Ala Val Gly Leu Arg Ala Met

115 120 125

Asp His Phe Leu Ala Val Asp Arg Asp Ala Leu Ser Ala Trp Ala Glu

130 135 140

Arg Gly Ser Leu Pro Val Val Ile Lys His Gln Ala Ser Ala Gly Thr

145 150 155 160

Asp Ser Val Thr Phe Cys Ala Asp Gln Gly Glu Leu Leu Tyr Ser Phe

165 170 175

Asp Gln Leu Phe Gly Thr Val Asn Gln Leu Gly Glu Arg Asn Asn Ala

180 185 190

Val Leu Ala Gln Arg Leu Leu Val Gly Pro Glu Tyr Ser Ile Asn Gly

195 200 205

Val Ser Gly His Gly Lys His Leu Ile Thr Glu Ile Trp Arg His Glu

210 215 220

Lys Leu Pro Ala Pro Asp Gly Gly Trp Ile Tyr Asp Arg Ala Val Leu

225 230 235 240

Phe Asp Pro Thr Ser Pro Glu Met Gln Glu Ile Val Arg Met Val His

245 250 255

Phe Val Leu Asp Ala Leu Gly Ile Arg Tyr Gly Ala Asn His Thr Glu

260 265 270

Leu Ile Val Thr Ala Asp Gly Pro Thr Leu Ile Glu Cys Ala Ser Arg

275 280 285

Leu Ser Gly Gly Ile Val Arg Pro Ala Ala Asn Tyr Ala Val Gly Ala

290 295 300

Ser Gln Leu Asp Leu Val Gly Lys Leu Val Arg Glu Gly Glu Ser Ala

305 310 315 320

Ile Asp Asp Ile Ala Gln Thr Trp Gln Thr Leu Arg Tyr Ala Leu Trp

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Gln Val Gln Phe Ile Ser Asn Gln Glu Gly Val Val Ala Arg Ser Ser

340 345 350

Tyr Asp Glu Leu Leu Lys Thr Leu Lys Ser Asn Ala Trp Leu Gln Arg

355 360 365

Ala Pro Lys Glu Gly Asp Thr Val Val Lys Thr Val Asp Leu Phe Ser

370 375 380

Ser Pro Gly Ile Val Phe Met Ser His Ala Asp Gly Asn Val Leu His

385 390 395 400

Asp Asp Tyr Arg Thr Val Arg Glu Val Glu Arg Thr Ser Arg Leu Phe

405 410 415

Ser Val Gln

<210> 5

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Met Thr Gln Ala Lys Glu Asn Ile Leu Val Tyr Val Asp Gly Tyr Ser

1 5 10 15

Ser Gly Ser Gln Leu Pro Thr Leu Val Ala Leu Ser Gly His Asn Cys

20 25 30

Val His Val Ser Ser Ser Ala Asn Pro Pro Glu Tyr Tyr Leu Arg Thr

35 40 45

Tyr His Lys Asp Glu Tyr Ile Ala His Phe Glu Tyr Gln Gly Asp Ile

50 55 60

Gln Ser Leu Ala Ser Ala Val Thr Gln Trp His Pro Ala Ala Val Leu

65 70 75 80

Arg Gly Thr Glu Ser Gly Val Ile Val Ala Asp Leu Leu Ala Ala Ala

85 90 95

Leu Gln Leu Gly Gly Asn Asp Pro Ser Thr Ser Leu Ala Arg Arg Asp

100 105 110

Lys Tyr Thr Met His Glu Ser Leu Lys Ala Val Gly Leu Arg Ala Met

115 120 125

Asp His Phe Leu Ala Val Asp Arg Asp Ala Leu Ser Ala Trp Ala Glu

130 135 140

Arg Gly Ser Leu Pro Val Val Ile Lys Pro Gln Ala Ser Ala Gly Thr

145 150 155 160

Asp Ser Trp Thr Phe Cys Ala Asp Gln Gly Glu Leu Leu Tyr Ser Phe

165 170 175

Asp Gln Leu Phe Gly Thr Val Asn Gln Leu Gly Glu Arg Asn Leu Ala

180 185 190

Val Leu Ala Gln Arg Leu Leu Val Gly Pro Glu Tyr Ser Ile Asn Ile

195 200 205

Val Ser Gly His Gly Lys His Leu Ile Thr Glu Ile Trp Arg His Glu

210 215 220

Lys Leu Pro Ala Pro Asp Gly His Trp Ile Tyr Asp Arg Ala Val Leu

225 230 235 240

Phe Asp Pro Thr Ser Pro Glu Met Gln Glu Ile Val Arg Tyr Pro His

245 250 255

Phe Val Leu Asp Ala Leu Gly Ile Arg Tyr Gly Ala Asn Thr Thr Glu

260 265 270

Leu Ile Val Thr Ala Asp Gly Pro Thr Leu Ile Glu Cys Phe Ser Arg

275 280 285

Leu Ser Gly Gly Leu His Arg Pro Ala Ala Asn Tyr Ala Val Gly Ala

290 295 300

Ser Gln Leu Asp Leu Val Gly Lys Leu Val Arg Glu Gly Glu Ser Ala

305 310 315 320

Ile Asp Asp Ile Leu Gln Thr Trp Asp Thr Leu Arg Tyr Ala Leu Trp

325 330 335

Gln Val Gln Phe Ile Ser Asn Gln Glu Gly Val Val Ala Arg Ser Ser

340 345 350

Tyr Asp Glu Leu Leu Lys Thr Leu Lys Ser Asn Ala Trp Leu Gln Arg

355 360 365

Ala Pro Lys Glu Gly Asp Thr Val Val Lys Thr Val Asp Leu Phe Ser

370 375 380

Ser Pro Gly Ile Val Phe Met Ser His Ala Asp Gly Asn Val Leu His

385 390 395 400

Asp Asp Tyr Arg Thr Val Arg Glu Val Glu Arg Thr Ser Arg Leu Phe

405 410 415

Ser Val Gln

<210> 6

<211> 403

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Met Arg Val Leu Val Ile Asn Ser Gly Ser Ser Ser Ile Lys Tyr Gln

1 5 10 15

Leu Ile Glu Met Glu Gly Glu Lys Val Leu Cys Lys Gly Ile Ala Glu

20 25 30

Arg Ile Gly Ile Glu Gly Ser Arg Leu Val His Arg Val Gly Asp Glu

35 40 45

Lys His Val Ile Glu Arg Glu Leu Pro Asp His Glu Glu Ala Leu Lys

50 55 60

Leu Ile Leu Asn Thr Leu Val Asp Glu Lys Leu Gly Val Ile Lys Asp

65 70 75 80

Leu Lys Glu Ile Asp Ala Val Gly His Arg Val Val His Gly Gly Glu

85 90 95

Arg Phe Lys Glu Ser Val Leu Val Asp Glu Glu Val Leu Lys Ala Ile

100 105 110

Glu Glu Val Ser Pro Leu Ala Pro Leu His Asn Pro Ala Asn Leu Met

115 120 125

Gly Ile Lys Ala Ala Met Lys Leu Leu Pro Gly Val Pro Asn Val Ala

130 135 140

Val Phe Asp Thr Ala Phe His Gln Thr Ile Pro Gln Lys Ala Tyr Leu

145 150 155 160

Tyr Ala Ile Pro Tyr Glu Tyr Tyr Glu Lys Tyr Lys Ile Arg Arg Tyr

165 170 175

Gly Phe His Gly Thr Ser His Arg Tyr Val Ser Lys Arg Ala Ala Glu

180 185 190

Ile Leu Gly Lys Lys Leu Glu Glu Leu Lys Ile Ile Thr Cys His Ile

195 200 205

Gly Asn Gly Ala Ser Val Ala Ala Val Lys Tyr Gly Lys Cys Val Asp

210 215 220

Thr Ser Met Gly Phe Thr Pro Leu Glu Gly Leu Val Met Gly Thr Arg

225 230 235 240

Ser Gly Asp Leu Asp Pro Ala Ile Pro Phe Phe Ile Met Glu Lys Glu

245 250 255

Gly Ile Ser Pro Gln Glu Met Tyr Asp Ile Leu Asn Lys Lys Ser Gly

260 265 270

Val Tyr Gly Leu Ser Lys Gly Phe Ser Ser Asp Met Arg Asp Ile Glu

275 280 285

Glu Ala Ala Leu Lys Gly Asp Glu Trp Cys Lys Leu Val Leu Glu Ile

290 295 300

Tyr Asp Tyr Arg Ile Ala Lys Tyr Ile Gly Ala Tyr Ala Ala Ala Met

305 310 315 320

Asn Gly Val Asp Ala Ile Val Phe Thr Ala Gly Val Gly Glu Asn Ser

325 330 335

Pro Ile Thr Arg Glu Asp Val Cys Ser Tyr Leu Glu Phe Leu Gly Val

340 345 350

Lys Leu Asp Lys Gln Lys Asn Glu Glu Thr Ile Arg Gly Lys Glu Gly

355 360 365

Ile Ile Ser Thr Pro Asp Ser Arg Val Lys Val Leu Val Val Pro Thr

370 375 380

Asn Glu Glu Leu Met Ile Ala Arg Asp Thr Lys Glu Ile Val Glu Lys

385 390 395 400

Ile Gly Arg

<210> 7

<211> 1194

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

atgggcgtga ccaaaacccc gctgtatgaa accctgaacg aaagcagcgc ggtggcgctg 60

gcggtgaaac tgggcctgtt tccgagcaaa agcaccctga cctgccagga aattggcgat 120

ggcaacctga actatgtgtt tcatatttat gatcaggaac atgatcgcgc gctgattatt 180

aaacaggcgg tgccgtatgc gaaagtggtg tgggaaagct ggccgctgac cattgatcgc 240

gcgcgcattg aaagcagcgc gctgattcgc cagggcgaac atgtgccgca tctggtgccg 300

cgcgtgtttt atagcgatac cgaaatggcg gtgaccgtga tggaagatct gagccatctg 360

aaaattgcgc gcaaaggcct gattgaaggc tataactatc cgcatctgag ccagcatatt 420

ggcgaatttc tgggcaaaac cctgttttat agcagcgatt atgcgctgga accgaaagtg 480

aaaaaacagc tggtgaaaca gtttaccaac ccggaactgt gcgatattac cgaacgcctg 540

gtgtttaccg atccgttttt tgatcatgat accaacgatt ttgaagaaga actgcgcccg 600

tttgtggaaa aactgtggaa caacgatagc gtgaaaattg aagcggcgaa actgaaaaaa 660

agctttctga ccagcgcgga aaccagcatt catggcgatc tgcataccgg cagcattttt 720

gcgagcgaac atgaaaccaa agtgatggat ccggaatttg cgttttatgg cccgattggc 780

tttgatgtgg gccagtttat tgcgaacctg tttctgaacg cgctgagccg cgatggcgcg 840

gatcgcgaac cgctgtatga acatgtgaac caggtgtggg aaacctttga agaaaccttt 900

agcgaagcgt ggcagaaaga tagcctggat gtgtatgcga acattgatgg ctatctgacc 960

gataccctga gccatatttt tgaagaagcg attggctttg cgggctgcga actgattcgc 1020

cgcaccattg gcctggcgca tgtggcggat ctggatacca ttgtgccgtt tgataaacgc 1080

attggccgca aacgcctggc gctggaaacc ggcaccgcgt ttattgaaaa acgcagcgaa 1140

tttaaaacca ttaccgatgt gattgaactg tttaaactgc tggtgaaaga atga 1194

<210> 8

<211> 834

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

atgagccagg tgcagtttag ccataacccg ctgttttgca tttggaaaat taaaacctat 60

aaaccggatt ttaccgtgcg cgtggcgttt attctgggca gcggcctgtt tgcgctggcg 120

gatcagattg aaaacgcggt ggcgattagc tatgaaaaac tgccgggctt tccggtgagc 180

accgtgcatg gcgcggcggg cgaactggaa gtgggccatc tgcagggcgt gccggtggtg 240

tgcatgaaag gccgcggcca tttttatacc ggccgcggca tgaccattat gaccgatgcg 300

attcgcaact ttaaactgct gggctgcgaa ctgctgtttt gcaccaacgc ggcgggcagc 360

ctgcgcccgg aacgcggcgc gggcagcctg gtggcgctga aagatcatat taacaccatg 420

ctgatgaccc cgatggtggg cctgaacgat gatcgctttg tggaacgctt ttttagcctg 480

gcgaacgcgg aagatgcgga atatattgcg ctgctgcaga aagtggcgaa agaagaaggc 540

tttccgctga ccccgagcgt gtttgtgagc tatccgggcc cgaactttga aaccgcggcg 600

gaaattcgca tgatgcagat tattggcggc gatgtggtgg gcatgagcgt ggtgccggaa 660

gtgattagcg cgcgccattg cgatctgaaa gtggtggcgg tgagcgcgat taccaacatg 720

gcggaaggcc tgagcgatgt gaaactgagc catgcgcaga ccctggcggc ggcggaactg 780

agcaaacaga actttattaa cctgatttgc ggctttctgc gcaaaattgc gtag 834

<210> 9

<211> 834

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

atgagccagg tgcagtttag ccataacccg ctgttttgca tttggaaaat taaaacctat 60

aaaccggatt ttacccagcg cgtggcgttt attctgggca gcggcctgtt tgcgctggcg 120

gatcagattg aaaacgcggt ggcgattagc tatgaaaaac tgccgggctt tccggtgagc 180

accgtgcatg gcgcggcggg cgaactggaa gtgggccatc tgcagggcgt gccggtggtg 240

tgcatgaaag gccgctgcgg cccgtatgaa ggccgcggca tgaccattat gaccgatgcg 300

attcgcacct ttaaactgct gggctgcgaa ctgctgtttt gcaccaacgc ggcgggcagc 360

ctgcgcccgg aagtgggcgc gtttagcctg gtggcgctga aagatcatat taacaccatg 420

ctgatgaccc cgatggtggg cctgaacgat attcgctttg gcgaacgctt ttttagcctg 480

gcgaacgcgg aagatgcgga atatcgcgcg gatctgcaga aagtggcgaa agaagaaggc 540

tttccgctga ccccgagcgt gtttgtgagc tatccgggcc cgaactttga aaccgcggcg 600

gaaattcgca tgatgcagat tattggcggc gatgtggtgg gcatgagcgt ggtgccggaa 660

gtgattagcg cgcgccattg cgatctgaaa gtggtggcgg tgagcgcgat taccaacatg 720

gcggaaggcc tgagcgatgt gaaactgagc catgcgcaga ccctggcggc ggcggaactg 780

agcaaacaga actttattaa cctgatttgc ggctttctgc gcaaaattgc gtag 834

<210> 10

<211> 1260

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

atgacccagg cgaaagaaaa cattctggtg tatgtggatg gctatagcag cggcagccag 60

ctgccgaccc tggtggcgct gagcggctgg aaatgcgtgc atgtgagcag cagcgcgaac 120

ccgccggaat attatctgcg cacctatcat aaagatgaat atattgcgca ttttgaatat 180

cagggcgata ttcagagcct ggcgagcgcg gtggaagcgt ggcatccggc ggcggtgctg 240

cgcggcagcg aaagcggcgt gattgtggcg gatctgctgg cggcggcgct ggatctgggc 300

ggcaacgatc cgagcaccag cctggcgcgc cgcgataaat ataccatgca tgaaagcctg 360

aaagcggtgg gcctgcgcgc gatggatcat tttctggcgg tggatcgcga tgcgctgagc 420

gcgtgggcgg aacgcggcag cctgccggtg gtgattaaac atcaggcgag cgcgggcacc 480

gatagcgtga ccttttgcgc ggatcagggc gaactgctgt atagctttga tcagctgttt 540

ggcaccgtga accagctggg cgaacgcaac aacgcggtgc tggcgcagcg cctgctggtg 600

ggcccggaat atagcattaa cggcgtgagc ggccatggca aacatctgat taccgaaatt 660

tggcgccatg aaaaactgcc ggcgccggat ggcggctgga tttatgatcg cgcggtgctg 720

tttgatccga ccagcccgga aatgcaggaa attgtgcgca tggtgcattt tgtgctggat 780

gcgctgggca ttcgctatgg cgcgaaccat accgaactga ttgtgaccgc ggatggcccg 840

accctgattg aatgcgcgag ccgcctgagc ggcggcattg tgcgcccggc ggcgaactat 900

gcggtgggcg cgagccagct ggatctggtg ggcaaactgg tgcgcgaagg cgaaagcgcg 960

attgatgata ttgcgcagac ctggcagacc ctgcgctatg cgctgtggca ggtgcagttt 1020

attagcaacc aggaaggcgt ggtggcgcgc agcagctatg atgaactgct gaaaaccctg 1080

aaaagcaacg cgtggctgca gcgcgcgccg aaagaaggcg ataccgtggt gaaaaccgtg 1140

gatctgttta gcagcccggg cattgtgttt atgagccatg cggatggcaa cgtgctgcat 1200

gatgattatc gcaccgtgcg cgaagtggaa cgcaccagcc gcctgtttag cgtgcagtga 1260

<210> 11

<211> 1260

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

atgacccagg cgaaagaaaa cattctggtg tatgtggatg gctatagcag cggcagccag 60

ctgccgaccc tggtggcgct gagcggccat aactgcgtgc atgtgagcag cagcgcgaac 120

ccgccggaat attatctgcg cacctatcat aaagatgaat atattgcgca ttttgaatat 180

cagggcgata ttcagagcct ggcgagcgcg gtgacccagt ggcatccggc ggcggtgctg 240

cgcggcaccg aaagcggcgt gattgtggcg gatctgctgg cggcggcgct gcagctgggc 300

ggcaacgatc cgagcaccag cctggcgcgc cgcgataaat ataccatgca tgaaagcctg 360

aaagcggtgg gcctgcgcgc gatggatcat tttctggcgg tggatcgcga tgcgctgagc 420

gcgtgggcgg aacgcggcag cctgccggtg gtgattaaac cgcaggcgag cgcgggcacc 480

gatagctgga ccttttgcgc ggatcagggc gaactgctgt atagctttga tcagctgttt 540

ggcaccgtga accagctggg cgaacgcaac ctggcggtgc tggcgcagcg cctgctggtg 600

ggcccggaat atagcattaa cattgtgagc ggccatggca aacatctgat taccgaaatt 660

tggcgccatg aaaaactgcc ggcgccggat ggccattgga tttatgatcg cgcggtgctg 720

tttgatccga ccagcccgga aatgcaggaa attgtgcgct atccgcattt tgtgctggat 780

gcgctgggca ttcgctatgg cgcgaacacc accgaactga ttgtgaccgc ggatggcccg 840

accctgattg aatgctttag ccgcctgagc ggcggcctgc atcgcccggc ggcgaactat 900

gcggtgggcg cgagccagct ggatctggtg ggcaaactgg tgcgcgaagg cgaaagcgcg 960

attgatgata ttctgcagac ctgggatacc ctgcgctatg cgctgtggca ggtgcagttt 1020

attagcaacc aggaaggcgt ggtggcgcgc agcagctatg atgaactgct gaaaaccctg 1080

aaaagcaacg cgtggctgca gcgcgcgccg aaagaaggcg ataccgtggt gaaaaccgtg 1140

gatctgttta gcagcccggg cattgtgttt atgagccatg cggatggcaa cgtgctgcat 1200

gatgattatc gcaccgtgcg cgaagtggaa cgcaccagcc gcctgtttag cgtgcagtga 1260

<210> 12

<211> 1212

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

atgcgcgtgc tggtgattaa cagcggcagc agcagcatta aatatcagct gattgaaatg 60

gaaggcgaaa aagtgctgtg caaaggcatt gcggaacgca ttggcattga aggcagccgc 120

ctggtgcatc gcgtgggcga tgaaaaacat gtgattgaac gcgaactgcc ggatcatgaa 180

gaagcgctga aactgattct gaacaccctg gtggatgaaa aactgggcgt gattaaagat 240

ctgaaagaaa ttgatgcggt gggccatcgc gtggtgcatg gcggcgaacg ctttaaagaa 300

agcgtgctgg tggatgaaga agtgctgaaa gcgattgaag aagtgagccc gctggcgccg 360

ctgcataacc cggcgaacct gatgggcatt aaagcggcga tgaaactgct gccgggcgtg 420

ccgaacgtgg cggtgtttga taccgcgttt catcagacca ttccgcagaa agcgtatctg 480

tatgcgattc cgtatgaata ttatgaaaaa tataaaattc gccgctatgg ctttcatggc 540

accagccatc gctatgtgag caaacgcgcg gcggaaattc tgggcaaaaa actggaagaa 600

ctgaaaatta ttacctgcca tattggcaac ggcgcgagcg tggcggcggt gaaatatggc 660

aaatgcgtgg ataccagcat gggctttacc ccgctggaag gcctggtgat gggcacccgc 720

agcggcgatc tggatccggc gattccgttt tttattatgg aaaaagaagg cattagcccg 780

caggaaatgt atgatattct gaacaaaaaa agcggcgtgt atggcctgag caaaggcttt 840

agcagcgata tgcgcgatat tgaagaagcg gcgctgaaag gcgatgaatg gtgcaaactg 900

gtgctggaaa tttatgatta tcgcattgcg aaatatattg gcgcgtatgc ggcggcgatg 960

aacggcgtgg atgcgattgt gtttaccgcg ggcgtgggcg aaaacagccc gattacccgc 1020

gaagatgtgt gcagctatct ggaatttctg ggcgtgaaac tggataaaca gaaaaacgaa 1080

gaaaccattc gcggcaaaga aggcattatt agcaccccgg atagccgcgt gaaagtgctg 1140

gtggtgccga ccaacgaaga actgatgatt gcgcgcgata ccaaagaaat tgtggaaaaa 1200

attggccgct ga 1212

Claims (3)

1. A method for preparing puromycin by an enzymatic method, which is characterized by comprising the following steps:

under the action of methyl thioribose kinase and ATP, the 3-amino-D-ribose generates beta-aminoribose-1-phosphate;

beta-aminoribose-1-phosphate and N, N-dimethyl purine generate 3-amino-N, N-dimethyl adenosine under the action of purine nucleoside phosphorylase;

3-amino-N, N-dimethyl adenosine and methyl tyrosine generate puromycin under the action of amino acid ligase and ATP;

the amino acid sequence of the methyl thioribose kinase is shown as SEQ ID No. 1:

MGVTKTPLYETLNESSAVALAVKLGLFPSKSTLTCQEIGDGNLNYVFHIYDQEHDRALIIKQAVPYAKVVWESWPLTIDRARIESSALIRQGEHVPHLVPRVFYSDTEMAVTVMEDLSHLKIARKGLIEGYNYPHLSQHIGEFLGKTLFYSSDYALEPKVKKQLVKQFTNPELCDITERLVFTDPFFDHDTNDFEEELRPFVEKLWNNDSVKIEAAKLKKSFLTSAETSIHGDLHTGSIFASEHETKVMDPEFAFYGPIGFDVGQFIANLFLNALSRDGADREPLYEHVNQVWETFEETFSEAWQKDSLDVYANIDGYLTDTLSHIFEEAIGFAGCELIRRTIGLAHVADLDTIVPFDKRIGRKRLALETGTAFIEKRSEFKTITDVIELFKLLVKE

the purine nucleoside phosphorylase PNP comprises PNP-a or PNP-b;

the amino acid sequence of PNP-a is shown as SEQ ID No. 2:

MSQVQFSHNPLFCIWKIKTYKPDFTVRVAFILGSGLFALADQIENAVAISYEKLPGFPVSTVHGAAGELEVGHLQGVPVVCMKGRGHFYTGRGMTIMTDAIRNFKLLGCELLFCTNAAGSLRPERGAGSLVALKDHINTMLMTPMVGLNDDRFVERFFSLANAEDAEYIALLQKVAKEEGFPLTPSVFVSYPGPNFETAAEIRMMQIIGGDVVGMSVVPEVISARHCDLKVVAVSAITNMAEGLSDVKLSHAQTLAAAELSKQNFINLICGFLRKIA

the amino acid sequence of PNP-b is shown as SEQ ID No. 3:

MSQVQFSHNPLFCIWKIKTYKPDFTQRVAFILGSGLFALADQIENAVAISYEKLPGFPVSTVHGAAGELEVGHLQGVPVVCMKGRCGPYEGRGMTIMTDAIRTFKLLGCELLFCTNAAGSLRPEVGAFSLVALKDHINTMLMTPMVGLNDIRFGERFFSLANAEDAEYRADLQKVAKEEGFPLTPSVFVSYPGPNFETAAEIRMMQIIGGDVVGMSVVPEVISARHCDLKVVAVSAITNMAEGLSDVKLSHAQTLAAAELSKQNFINLICGFLRKIA

the amino acid ligase aaLigase comprises aaLigase-1 or aaLigase-2;

the amino acid sequence of the aaligase-1 is shown in SEQ ID No. 4:

MTQAKENILVYVDGYSSGSQLPTLVALSGWKCVHVSSSANPPEYYLRTYHKDEYIAHFEYQGDIQSLASAVEAWHPAAVLRGSESGVIVADLLAAALDLGGNDPSTSLARRDKYTMHESLKAVGLRAMDHFLAVDRDALSAWAERGSLPVVIKHQASAGTDSVTFCADQGELLYSFDQLFGTVNQLGERNNAVLAQRLLVGPEYSINGVSGHGKHLITEIWRHEKLPAPDGGWIYDRAVLFDPTSPEMQEIVRMVHFVLDALGIRYGANHTELIVTADGPTLIECASRLSGGIVRPAANYAVGASQLDLVGKLVREGESAIDDIAQTWQTLRYALWQVQFISNQEGVVARSSYDELLKTLKSNAWLQRAPKEGDTVVKTVDLFSSPGIVFMSHADGNVLHDDYRTVREVERTSRLFSVQ

the amino acid sequence of the aaligase-2 is shown in SEQ ID No. 5:

MTQAKENILVYVDGYSSGSQLPTLVALSGHNCVHVSSSANPPEYYLRTYHKDEYIAHFEYQGDIQSLASAVTQWHPAAVLRGTESGVIVADLLAAALQLGGNDPSTSLARRDKYTMHESLKAVGLRAMDHFLAVDRDALSAWAERGSLPVVIKPQASAGTDSWTFCADQGELLYSFDQLFGTVNQLGERNLAVLAQRLLVGPEYSINIVSGHGKHLITEIWRHEKLPAPDGHWIYDRAVLFDPTSPEMQEIVRYPHFVLDALGIRYGANTTELIVTADGPTLIECFSRLSGGLHRPAANYAVGASQLDLVGKLVREGESAIDDILQTWDTLRYALWQVQFISNQEGVVARSSYDELLKTLKSNAWLQRAPKEGDTVVKTVDLFSSPGIVFMSHADGNVLHDDYRTVREVERTSRLFSVQ

the reaction system with ATP also comprises an ATP circulating system composed of acetate kinase and acetyl phosphate;

the amino acid sequence of the acetate kinase is shown in SEQ ID No. 6:

MRVLVINSGSSSIKYQLIEMEGEKVLCKGIAERIGIEGSRLVHRVGDEKHVIERELPDHEEALKLILNTLVDEKLGVIKDLKEIDAVGHRVVHGGERFKESVLVDEEVLKAIEEVSPLAPLHNPANLMGIKAAMKLLPGVPNVAVFDTAFHQTIPQKAYLYAIPYEYYEKYKIRRYGFHGTSHRYVSKRAAEILGKKLEELKIITCHIGNGASVAAVKYGKCVDTSMGFTPLEGLVMGTRSGDLDPAIPFFIMEKEGISPQEMYDILNKKSGVYGLSKGFSSDMRDIEEAALKGDEWCKLVLEIYDYRIAKYIGAYAAAMNGVDAIVFTAGVGENSPITREDVCSYLEFLGVKLDKQKNEETIRGKEGIISTPDSRVKVLVVPTNEELMIARDTKEIVEKIGR；

the method is a multi-component one-pot method;

the multi-component one-pot reaction system also comprises an activator and a substrate cosolvent;

the activator is magnesium chloride, and the substrate cosolvent is isopropanol.

2. The method according to claim 1, wherein the ratio of 3-amino-D-ribose, N-dimethylpurine, methyltyrosine, ATP, acetyl phosphate, activator, substrate co-solvent, methylthioribose kinase, purine nucleoside phosphorylase, amino acid ligase, acetate kinase in the multicomponent one pot reaction system is (50-150) mM: (50-150) mM: (50-150) mM: (1-2) mM: (100-300) mM: (5-15) mM: (100-150) mL: (1000-1500) U: (800-1500) U: (800-1500) U: (2000-4000) U.

3. The method of claim 1, wherein the reaction conditions of the multicomponent one pot process are: the pH is 7.0-8.5, and the mixture is stirred for 2-10 hours at 15-30 ℃.