Cytochrome P450BM3 mutant and application thereof in synthesis of trenbolone acetate

文档序号：128342 发布日期：2021-10-22 浏览：50次中文

阅读说明：本技术 一种细胞色素p450 bm3突变体及其在醋酸群勃龙合成中的应用 (Cytochrome P450BM3 mutant and application thereof in synthesis of trenbolone acetate ) 是由李爱涛彭雅琴高成华赵晶张铮斌李纯于 2021-06-28 设计创作，主要内容包括：本发明公开了一种细胞色素P450BM3突变体及其在醋酸群勃龙合成中的应用,所述细胞色素P450BM3突变体选自：突变体LG-23/T438S,LG-23/T438A和/或LG-23/T438G,氨基酸序列如SEQ ID NO：1,SEQ ID NO:2和SEQ ID NO:3所示,其都是在突变体LG-23的基础上定点突变得到。经验证所述细胞色素P450BM3突变体和LG-23都具有甾体化合物11α羟基化催化活性,可应用于醋酸群勃龙的合成中。使用BM3突变体酶和17β-HSD酶一步法催化4,9物生成11α-羟基甲基双烯醇酮,并结合化学催化生成醋酸群勃龙。本发明提供了一种生物酶催化和化学催化结合合成醋酸群勃龙的方法,不仅简化药物合成步骤,提高催化选择性,减少副产物提高产率,并且反应条件温和,成本低廉,绿色环保高效,对推动我国甾体药物的开发进程有着重要的应用价值。(The invention discloses a cytochrome P450BM3 mutant and application thereof in synthesis of trenbolone acetate, wherein the cytochrome P450BM3 mutant is selected from the following components: the mutants LG-23/T438S, LG-23/T438A and/or LG-23/T438G have amino acid sequences shown in SEQ ID NO:1, SEQ ID NO 2 and SEQ ID NO 3, which are obtained by site-directed mutagenesis on the basis of the mutant LG-23. The cytochrome P450BM3 mutant and LG-23 are proved to have catalytic activity of steroid 11 alpha hydroxylation and can be applied to synthesis of trenbolone acetate. BM3 mutant enzyme and 17 beta-HSD enzyme are used to catalyze the 4,9 product to generate 11 alpha-hydroxymethyl dienolone in one step, and the trenbolone acetate is generated by combining chemical catalysis. The invention provides a method for synthesizing trenbolone acetate by combining biological enzyme catalysis and chemical catalysis, which simplifies the drug synthesis steps, improves the catalytic selectivity, reduces byproducts, improves the yield, has mild reaction conditions, low cost, environmental protection and high efficiency, and has important application value for promoting the development process of steroid drugs in China.)

1. A cytochrome P450BM3 mutant, wherein the cytochrome P450BM3 mutant is selected from the group consisting of: mutant LG-23/T438S, LG-23/T438A and/or LG-23/T438G, the amino acid sequence of the mutant LG-23/T438S is shown in SEQ ID NO:1, the amino acid sequence of the mutant LG-23/T438A is shown as SEQ ID NO. 2, and the amino acid sequence of the mutant LG-23/T438G is shown as SEQ ID NO. 3.

2. The cytochrome P450BM3 mutant according to claim 1, wherein the mutants LG-23/T438S, LG-23/T438A and LG-23/T438G are mutants LG-23, wherein the mutation of threonine at position 438 to serine, alanine and glycine, respectively.

3. A gene encoding the cytochrome P450BM3 mutant as claimed in claim 1 wherein the nucleotide sequence encoding the mutant LG-23/T438S gene is as shown in SEQ ID NO:4, the nucleotide sequence of the gene for coding the mutant LG-23/T438A is shown as SEQ ID NO. 5, and the nucleotide sequence of the gene for coding the mutant LG-23/T438G is shown as SEQ ID NO. 6.

4. A vector comprising the gene encoding the cytochrome P450BM3 mutant according to claim 3.

5. A cell comprising the vector of claim 4.

6. The cytochrome P450BM3 mutant as claimed in claim 1, or the mutant LG-23 as claimed in claim 2, or the vector as claimed in claim 4, or the cell as claimed in claim 5 for use in catalysing the alpha hydroxylation of steroid 11 alpha.

7. Use of a cytochrome P450BM3 mutant as defined in claim 1 or a mutant LG-23 as defined in claim 2 or a vector as defined in claim 4 or a cell as defined in claim 5 in the synthesis of trenbolone acetate.

8. A synthetic method of trenbolone acetate is characterized by comprising the following steps:

step 1, generating 11 alpha-hydroxymethyl dienolone by using estra-4, 9-diene-3, 17-dione under the action of a cytochrome P450BM3 mutant as claimed in claim 1 or a mutant LG-23 as claimed in claim 2, and 17 beta hydroxyl dehydrogenase and glucose dehydrogenase;

step 2, reacting the 11 alpha-hydroxymethyl dienolone with p-toluenesulfonic acid monohydrate, and extracting with trichloromethane to obtain trenbolone;

and 3, carrying out esterification reaction on the trenbolone, 4-dimethylaminopyridine and acetic anhydride to obtain the trenbolone acetate.

9. The synthesis method according to claim 8, wherein step 1 specifically comprises: dissolving genetic engineering bacteria co-expressing or respectively expressing cytochrome P450BM3 mutant and 17 beta hydroxyl dehydrogenase in buffer solution, adding glucose dehydrogenase, estra-4, 9-diene-3, 17-dione as substrate, and NADP as cofactor⁺And glucose, reacting completely at 20-30 ℃, adding ethyl acetate to extract reaction liquid to obtain ethyl acetate extract, dehydrating with anhydrous sodium sulfate, filtering, and concentrating under reduced pressure to obtain the crude product of 11 alpha-hydroxymethyl dienolone.

10. The synthesis method according to claim 8, wherein the step 2 is specifically: dissolving the 11 alpha-hydroxymethyl dienolone crude product in chloroform, adding p-toluenesulfonic acid monohydrate, stirring at room temperature until the reaction is complete, adding saturated sodium carbonate to stop the reaction, extracting the reaction solution with chloroform to obtain a chloroform extract, dehydrating with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, separating and purifying by column chromatography, concentrating and drying to obtain the trenbolone.

Technical Field

The invention belongs to the technical field of biocatalysis enzyme, and particularly relates to a cytochrome P450BM3 mutant and application thereof in synthesis of trenbolone acetate.

Background

Steroid hormone drugs are the second class of drugs second to antibiotics in the world, and are widely applied to the treatment of inflammation, cardiovascular diseases, tumors, skin diseases, endocrine disorders, senile diseases and other diseases. The natural steroid resources in China are rich, the production scale and the product quality are close to the advanced level of the world, but the research and development technical level of the steroid drugs still has certain gap, mainly because the steroid drugs have complicated synthesis steps, complex reaction, obvious group remote effect, low yield and particularly difficult separation and purification.

Steroid hormones can be divided into three main groups according to their physiological activities: anabolic hormones, adrenocortical hormones, and sex hormones. The protein assimilation hormone is commonly called synthetic hormone, is a steroid hormone artificially synthesized by pseudoandrogen, reduces the androgen activity and improves the protein isoactivity by carrying out structural modification on the androgen, and has the advantages of easy absorption after administration, high blood concentration, large in vivo activity and multiple functions. Trenbolone acetate is one of protein assimilation elements, is widely applied clinically, and can rapidly increase muscle strength and circumference and improve muscle quality. In addition, trenbolone acetate can prevent mad cow disease, and the market demand at home and abroad is increasing day by day.

The traditional production process of trenbolone acetate takes 4, 9(11) -dien androstane 3-17-diketone as a raw material, takes p-toluenesulfonic acid or acetyl chloride as a catalyst, protects 3-carbonyl, and then obtains trenbolone by reduction, dehydrogenation and hydrolysis; finally, trenbolone and acetic anhydride are subjected to esterification reaction to obtain trenbolone acetate. However, the route has many byproducts, low quality and low yield, and is not favorable for large-scale production and application.

The prior art discloses a method for preparing trenbolone acetate by taking 4,9 (estra-4, 9-diene-3, 17-dione, CAS: 5173-46-6) as a raw material and carrying out dimethyl etherification at the 3-position, reduction at the 17-position, hydrolysis at the 3-position, dehydrogenation at the 11-position and 12-position and esterification at the 17-position in 5 steps, however, acetyl chloride used in the production line has strong irritation and corrosivity, acetic acid is generated after the reaction is stopped by adding water, waste water is difficult to treat, and the subsequent reduction hydrolysis reaction is solvent replacement after the reaction is stopped, multiple times of water washing, complex operation, large amount of waste water and low yield, and the total yield is 63.1%.

Patent CN108017682A discloses a synthetic method of trenbolone acetate, which can obtain trenbolone acetate by dehydrogenation and esterification based on the prior art, and a new synthetic route of trenbolone acetate is obtained by optimizing reaction route and conditions. However, the chemical synthesis route adopted by the patent still has the problems of complicated steps, low efficiency, complex reaction, more byproducts, difficult purification and separation and the like, and also generates a large amount of pollution wastes. Therefore, how to improve the reaction selectivity, reduce the byproducts, maintain mild reaction conditions and low cost is an important problem to be solved urgently at present.

Disclosure of Invention

The invention provides a P450BM3 mutant enzyme with 11 alpha hydroxylation on estra-4, 9-diene-3, 17-dione (4,9 products) and methyldienol ketone, which uses P450BM3 mutant enzyme and 17 beta hydroxyl dehydrogenase (17 beta-HSD enzyme) to catalyze the 4,9 products to generate 11 alpha-hydroxymethyl dienol ketone by one step from cheap and easily available 4,9 products, and uses GDH enzyme to regenerate and circulate cofactor NADPH, and further combines chemical catalysis reaction to generate trenbolone acetate. The method has the advantages of high selectivity of bio-enzyme catalysis, few byproducts, mild reaction conditions, low cost, environmental protection and high efficiency, and can synthesize trenbolone acetate more efficiently and quickly by combining bio-enzyme catalysis and chemical catalysis.

In order to achieve the purpose, the invention adopts the technical scheme that:

one of the objectives of the present invention is to provide a cytochrome P450BM3 mutant, wherein the cytochrome P450BM3 mutant is selected from the group consisting of: mutant LG-23/T438S, LG-23/T438A and/or LG-23/T438G, the amino acid sequence of the mutant LG-23/T438S is shown in SEQ ID NO:1, the amino acid sequence of the mutant LG-23/T438A is shown as SEQ ID NO. 2, and the amino acid sequence of the mutant LG-23/T438G is shown as SEQ ID NO. 3.

Further, the mutants LG-23/T438S, LG-23/T438A and LG-23/T438G are characterized in that threonine at position 438 is mutated into serine, alanine and glycine respectively on the basis of the mutant LG-23.

The invention also aims to provide a gene for coding the cytochrome P450BM3 mutant, wherein the nucleotide sequence of the mutant LG-23/T438S gene is shown as SEQ ID NO:4, the nucleotide sequence of the mutant LG-23/T438A gene is shown as SEQ ID NO. 5, and the nucleotide sequence of the mutant LG-23/T438G gene is shown as SEQ ID NO. 6.

The present invention also provides a vector comprising the gene of the cytochrome P450BM3 mutant.

The fourth object of the present invention is to provide a cell comprising the above vector.

The fifth purpose of the invention is to provide the cytochrome P450BM3 mutant, or the mutant LG-23, or the carrier, or the cell in the application of catalyzing the alpha hydroxylation of the steroid compound 11 alpha.

Further, the steroid compounds include, but are not limited to, estra-4, 9-diene-3, 17-dione and/or methyldienolone.

The sixth purpose of the invention is to provide the cytochrome P450BM3 mutant, the mutant LG-23, the vector or the application of the cell in the synthesis of trenbolone acetate.

The seventh purpose of the invention is to provide a method for synthesizing trenbolone acetate, which comprises the following steps:

step 1, generating 11 alpha-hydroxymethyl dienolone by using estra-4, 9-diene-3, 17-dione under the action of the cytochrome P450BM3 mutant or the mutant LG-23, and 17 beta hydroxyl dehydrogenase and glucose dehydrogenase;

step 2, reacting the 11 alpha-hydroxymethyl dienolone with p-toluenesulfonic acid monohydrate, and extracting with trichloromethane to obtain trenbolone;

and 3, carrying out esterification reaction on the trenbolone, 4-dimethylaminopyridine and acetic anhydride to obtain the trenbolone acetate.

Further, step 1 specifically comprises: dissolving genetic engineering bacteria co-expressing or respectively expressing cytochrome P450BM3 mutant and 17 beta hydroxyl dehydrogenase in buffer solution, adding glucose dehydrogenase, estra-4, 9-diene-3, 17-dione as substrate, and NADP as cofactor⁺And glucose, reacting completely at 20-30 ℃, adding ethyl acetate to extract reaction liquid to obtain ethyl acetate extract, dehydrating with anhydrous sodium sulfate, filtering, and concentrating under reduced pressure to obtain the crude product of 11 alpha-hydroxymethyl dienolone.

Furthermore, in step 1, the coding gene of cytochrome P450BM3 mutant and the coding gene of 17 beta hydroxyl dehydrogenase are operably connected with an expression regulatory sequence through RBS sequences in the sequence from front to back to obtain an expression vector co-expressed by the two enzyme genes.

Further, the co-expressed expression vector is transferred into escherichia coli to obtain the co-expressed genetic engineering bacteria.

Furthermore, in the step 1, a cosolvent Dimethylformamide (DMF) is used for preparing a mother solution of the estra-4, 9-diene-3, 17-diketone substrate.

Further, the OD of the co-expressed genetically engineered bacterium₆₀₀20-30, the mole concentration of estra-4, 9-diene-3, 17-dione is 1mM, the DMF content is 1% (v/v), NADP⁺The molar concentration is 0.5mM, the final concentration of the glucose dehydrogenase activity is 1U/mL, and the glucose content is 5% (m/v).

Furthermore, the glucose dehydrogenase encoding gene is independently connected with an expression regulatory sequence to obtain an expression vector of the glucose dehydrogenase gene.

Further, step 2 specifically comprises: dissolving the 11 alpha-hydroxymethyl dienolone crude product in chloroform, adding p-toluenesulfonic acid monohydrate, stirring at room temperature until the reaction is complete, adding saturated sodium carbonate to stop the reaction, extracting the reaction solution with chloroform to obtain a chloroform extract, dehydrating with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, separating and purifying by column chromatography, concentrating and drying to obtain the trenbolone.

Further, in step 2, 11 α -hydroxymethyl diketene: trichloromethane: the mass ratio of the p-toluenesulfonic acid monohydrate is 1: 25-35: 0.6-0.8.

Further, step 3 specifically comprises: dissolving trenbolone in dichloromethane, adding 4-Dimethylaminopyridine (DMAP) and acetic anhydride, controlling the temperature to be 20-25 ℃, stirring and reacting until the reaction is complete, adding a sodium carbonate solution, washing until the pH value is 8-9, and extracting a water phase by using dichloromethane; and combining all dichloromethane, dehydrating with anhydrous sodium sulfate, filtering, concentrating under reduced pressure until no distillate exists, adding isopropyl ether to carry out distillation on residual dichloromethane, adding isopropyl ether after all dichloromethane is distilled, cooling, crystallizing, centrifuging, and drying to obtain the trenbolone acetate.

Further, trenbolone: dichloromethane: DMAP: the mass ratio of acetic anhydride is 1: 15-25: 0.01-0.1: 0.8 to 1.2.

Compared with the prior art, the invention has the beneficial effects that: the invention provides a P450BM3 mutant enzyme for the first time: mutants LG-23/T438S, LG-23/T438A and LG-23/T438G are obtained by respectively mutating threonine at position 438 into serine, alanine and glycine on the basis of the mutant LG-23, and proved that the mutants LG-23/T438S, LG-23/T438A, LG-23/T438G and LG-23 all have the function of catalyzing the hydroxylation of a steroid compound 11 alpha, can be applied to the synthesis of trenbolone acetate, and from cheap and easily available 4 and 9, 11 alpha-hydroxymethyl dienolone is generated by using P450BM3 mutant enzyme and 17 beta-HSD enzyme through one-step catalysis of the 4 and 9 products, and GDH enzyme is used for regeneration and circulation of cofactor NADPH, and further combined with chemical catalysis reaction to generate the trenbolone acetate. The invention provides a method for synthesizing trenbolone acetate by combining biological enzyme catalysis and chemical catalysis, which simplifies the synthesis steps of the drugs, remarkably improves the catalytic selectivity, reduces byproducts, improves the yield, has mild reaction conditions, low cost, environmental protection and high efficiency, and has important production and application values for promoting the development process of steroid drugs in China.

Drawings

FIG. 1 is a graph showing the results of HPLC analysis of 11. alpha. hydroxylation of 4, 9-substance or methyldiketene dimer by mutants LG-23 and LG-23/T438S in example 1 of the present invention;

FIG. 2 shows the reaction process and detection results of the transformation of the 4, 9-compound into 11 α -hydroxymethyl diketene by the whole-cell catalysis of Escherichia coli with co-expression of P450BM 3-LG-23/T438S and 17 β -HSDcl in example 2 of the present invention;

FIG. 3 is a flow chart of the reaction of combining biological enzyme catalysis and chemical catalysis to produce trenbolone acetate in example 3 of the present invention;

FIG. 4 is an LC/MS analytical spectrum of 11 α -hydroxymethyldiketene prepared in example 3 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 site-directed mutagenesis and functional validation of P450BM3 enzyme mutant LG-23

1. Site-directed mutagenesis of mutant LG-23

This example shows that, on the basis of the P450BM3 enzyme mutant LG-23(Li A, Acevedo-Rocha C G, D' Amore L, et al, Regio-and Stereoselective Steroid hydrolysis at the C7-Position by Cytochrome P450 Monooxogenes Mutants [ J ]. Angewandte chemical Edition,2020) having Steroid 7 β hydroxylating activity, a plasmid vector pRSFDuet-LG-23 was used as a template, the gene of the mutant LG-23 was subjected to site-directed mutagenesis by using a primer PCR mutagenesis technique, and the 438 th threonine was mutated into serine, alanine and glycine, respectively, and a pair of primers were designed for the mutation sites, wherein the plasmid vector SFpRDuet-LG-23 is the existing unit.

In this example, the mutant LG-23/T438S is taken as an example, and a mutation primer (shown in SEQ ID NO:7-8 in the sequence Listing) which can be used for obtaining the mutant LG-23/T438S by site-directed mutagenesis is provided:

an upstream primer: 5'-TTAAAGAAACTTTATCTTTAAAACCTGAAGGCTTTG-3' the flow of the air in the air conditioner,

a downstream primer: 5'-ATTTCCGGCGTGTGAATCAAGCG-3' the flow of the air in the air conditioner,

wherein the bold sequence is a mutation site.

The PCR system (20. mu.L) was: 0.1-1 ng of template, 1. mu.L (10. mu.M) of each pair of mutation primers, 5. mu.L of Prime STAR Max DNA polymerase, and 20. mu.L of sterilized distilled water.

The PCR reaction program is: (1) pre-denaturation at 98 ℃ for 3 min; (2) denaturation at 98 ℃ for 10sec, (3) annealing at 56 ℃ for 15sec, (4) extension at 72 ℃ for 70sec, 28 cycles of steps (2) - (4) in total, final extension at 72 ℃ for 5min, and preservation at 8 ℃.

And detecting the PCR product obtained by amplification by using 0.7% agarose gel electrophoresis, and observing a band with the length equivalent to that of the P450BM3 gene, thereby judging that the gene for encoding the target mutant is amplified, so that the restriction enzyme Dpn I is directly added into the PCR product, the PCR product is digested at 37 ℃ for 3-5 h and then transformed into E.coli DH5 alpha competent cells, and the E.coli DH5 alpha competent cells are added with a culture medium for 1h for recovery and then uniformly coated on a solid LB plate containing 50 mu l/mg kanamycin. After overnight culture at 37 ℃, single clones were picked up to 3mL LB liquid medium containing 50. mu.l/mg kanamycin for culture, and then sent to a sequencer for sequencing to obtain the correct mutant, which was named LG-23/T438S and the mutation site of the coding sequence thereof included: R47W/S72W/F77Y/V78L/F81I/A82L/F87G/T88S/M177T/M185Q/I209T/A328G/A330W/T438S, wherein the amino acid sequence of the mutant LG-23/T438S is shown as a sequence table SEQ ID NO. 1, and the nucleotide sequence is shown as a sequence table SEQ ID NO. 4.

Respectively designing mutation primers by adopting a method similar to the method, and mutating the 438 th threonine to alanine to obtain a mutant LG-23/T438A, wherein the amino acid sequence of the mutant is shown as a sequence table SEQ ID NO. 2, and the nucleotide sequence of the mutant is shown as a sequence table SEQ ID NO. 5; the 438 th threonine is mutated into glycine to obtain a mutant LG-23/T438G, the amino acid sequence of which is shown as the sequence table SEQ ID NO. 3, and the nucleotide sequence of which is shown as the sequence table SEQ ID NO. 6.

2. Functional verification

E.coli BL21 competent cells were transformed with the plasmids pRSFDuet-LG-23/T438S, pRSFDuet-LG-23/T438A and pRSFDuet-LG-23/T438G obtained above, respectively, and then applied uniformly to a solid LB plate containing 50. mu.l/mg kanamycin after adding a medium for 1 hour to recover, and E.coli BL21 glycerol pipefish containing LG-23 stored at-80 ℃ was streaked on a solid LB plate containing 50. mu.l/mg kanamycin and cultured overnight at 37 ℃. Selecting single clone to 2mL liquid LB culture medium containing 50 mul/mg kanamycin, shaking and culturing at 37 ℃ overnight, taking 500 mul bacterial liquid to insert into 100mL triangular flask containing 50mL TB culture medium, placing in 37 ℃, shaking and culturing at 220rpm shaking table, and when the absorbance OD of the culture solution is₆₀₀When the concentration reaches 0.8, IPTG with the final concentration of 0.2mM is added for induction expression, the induction temperature is 25 ℃, and the induction lasts for 16-20 hours. The culture broth was centrifuged at 4000rpm at 4 ℃ for 10min, the cells were collected and washed once with 100mM potassium phosphate buffer (pH8.0), and the cells were stored at-80 ℃.

The cells were treated with 10mL of 100m M potassium phosphate buffer (containing 5% glucose, 5% glycerol, 0.5mM NADP) at pH8.0⁺10U GDH) were resuspended in 50mL centrifuge tubes and immediately snap frozen in liquid nitrogen. Then, the mixture was thawed in water at room temperature, and 5mL of the suspension was put in a 50mL Erlenmeyer flask, 50uL of the steroid substrate mother liquor (100 mM mother liquor prepared from DMF) was added thereto, and the mixture was reacted at 25 ℃ and 220rpm for 5 hours. Sampling at intervals, extracting the reaction solution with ethyl acetate with the same volume, centrifuging at a high speed for 3min, transferring the upper layer of ethyl acetate into a clean EP tube, resuspending the reaction solution with acetonitrile with the same volume after complete volatilization, filtering the reaction solution into a sample injection bottle by using a filter membrane with the diameter of 0.22 mu m, and detecting the conversion rate and the product distribution of the reaction by adopting HPLC.

The column was a ZORBAX SB C18 (250X 4.6mm) column, the mobile phase was acetonitrile/ultrapure water: 2min (10:90), 2-15 min (70:20), 15-17 min (10: 90); the column temperature was 40 ℃, the flow rate was 1.5mL/min, and the sample size was 10. mu.L. The ultraviolet detection wavelengths of the methyl dienolone, the estra-4, 9-diene-3, 17-dione (4,9 substance) and the hydroxylation product are 310 nm.

The data of the P450BM3 mutants LG-23, LG-23/T438S, LG-23/T438A and LG-23/T438G catalyzing the hydroxylation of 4,9 or methyldienolone 11 alpha at a concentration of 1mM (0.27g/L) are shown in Table 1, and the HPLC analysis results are shown in FIG. 1.

TABLE 1 cytochrome P450BM3 mutants catalyze steroid 11 alpha hydroxylation

According to the figure 1, the equations of P450BM3 mutant LG-23, LG-23/T438S, LG-23/T438S, LG-23/T438A or LG-23/T438G for catalyzing the 11 alpha hydroxylation of 4, 9-compound or methyl dienolone are shown as (1) and (2), respectively.

According to the results in Table 1, the 11 alpha hydroxylation selectivity of mutants LG-23/T438S, LG-23/T438A and LG-23/T438G to methyl dienolone is improved relative to the 11 alpha hydroxylation selectivity of the mutant LG-23 to methyl dienolone of 78%, and the 11 alpha hydroxylation selectivity of the mutant LG-23/T438S to methyl dienolone is improved to 92%. For the 4,9 mutant LG-23/T438S was also slightly more selective than LG-23 for 11 α hydroxylation. The result shows that the mutant LG-23/T438S has relatively better effect of catalyzing steroid 11 alpha hydroxylation, and the mutant LG-23/T438S is taken as an example for the synthesis of trenbolone acetate.

Example 2 construction of Co-expression recombinant plasmid and preparation of GDH crude enzyme lyophilized powder

Although the mutants LG-23/T438S or LG-23 can be directly used for carrying out 11 alpha hydroxylation on methyl dienolone to generate 11 alpha-hydroxymethyl dienolone, and then the trenbolone acetate is synthesized by a two-step chemical method. However, considering that the raw materials 4 and 9 are cheaper, 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) capable of reducing steroid 17 site ketone into hydroxyl is combined, and from cheaper and easily available 4 and 9, the mutant LG-23/T438S and 17 beta-HSD enzyme are used for catalyzing the 4 and 9 to generate 11 alpha-hydroxymethyl dienolone in one step, and glucose dehydrogenase is used for regenerating and recycling the cofactor NADPH.

Wherein the 17 beta-HSD is a steroid 17 beta hydroxydehydrogenase 17 beta-HSDcl enzyme from curvularia lunata (Cochliobolus lunatus), and the catalytic equation is shown as (3) or (4):

the glucose dehydrogenase is a glucose dehydrogenase (GDH enzyme) from bacillus megaterium (Priestia megaterium) and its catalytic equation is shown in (5):

(5) glucose + NADP⁺→ gluconolactone + NADPH

In order to catalyze 4, 9-substance one-step method to produce 11 alpha-hydroxymethyl dienolone, the genes of the three enzymes can be respectively cloned and expressed in a host; the mutant LG-23/T438S enzyme gene and the 17 beta-HSDcl enzyme gene can also be transferred into the same host cell to be co-expressed, and the single cell system is utilized to realize the catalysis and the transformation of the substrate; and the GDH enzyme gene is independently expressed, and the regeneration and circulation of the cofactor NADPH are realized by using the crude enzyme catalysis of the broken bacteria supernatant.

1. Construction method of recombinant expression vector for co-expressing LG-23/T438S enzyme and 17 beta-HSDcl enzyme

The gene encoding 17 β -HSDcl enzyme and the plasmid vector pRSFDuet-LG-23/T438S obtained in example 1 were PCR amplified using a primer containing a 20bp homologous end, in which the RBS sequence was located, to thereby linearize it. The 17 β -HSDcl gene and the linearized vector were then ligated together by forming a 20bp cohesive end by the action of T5 exonuclease. Primers containing homologous ends were first designed.

The linearized vector pRSFDuet-LG-23/T438S primer is (SEQ ID NO: 9-10):

5'-TAACCTAGGCTGCTGCCACCGC-3' as upstream primer;

5'-GATATATCTCCTTAGGTACCTTACCCAGCCCACACGTC-3' as downstream primer;

obtaining a linearization vector pRSFDuet-LG-23/T438S by adopting conventional PCR amplification;

the 17 beta-HSDcl enzyme gene fragment amplification primer is (SEQ ID NO: 11-12):

an upstream primer: 5'-TGGTACCTAAGGAGATATATCATGCCGCACGTGGAGAAC-3', respectively;

a downstream primer: 5'-GGTGGCAGCAGCCTAGGTTATTAGGCGGCGCCGCCGTC-3', respectively;

obtaining the 17 beta-HSDcl enzyme gene fragment by adopting the conventional PCR amplification.

Confirming the size and purity of the PCR product fragment by agarose gel electrophoresis, recovering the target nucleic acid fragment by agarose gel electrophoresis, and detecting the concentration and purity of the recovered nucleic acid sample by a micro-spectrophotometer. The method comprises the following steps of utilizing T5 exonuclease to connect and recover a 17 beta-HSDcl enzyme gene fragment and a plasmid vector fragment, and specifically comprising the following steps: adding the target fragment and a linearized vector (the amount of the linearized vector is controlled to be 30-50 ng) into a 5-mu-L reaction system according to a molar ratio of 3:1, then adding 0.5 mu L T5 exonuclease and 1 mu L NE buffer, and adding sterilized distilled water to supplement to 5 mu L. After T5 exonuclease is added, the cell is strictly timed for 5min, and after the time is up, 50. mu.L of E.coli DH5 alpha competent cells are immediately added to carry out transformation according to the basic steps of conventional transformation, and after the cell is added and recovered for 1h, the cell is evenly coated on a solid LB plate containing 50. mu.l/mg kanamycin. After overnight culture at 37 ℃, single clones were picked up to 3mL LB liquid medium containing 50 μ l/mg kanamycin for culture, and then sent to sequencing company for sequencing to obtain the correct recombinant plasmid, which was named: pRSFDuet-LG-23/T438S-17 β -HSDcl.

2. Functional verification of Co-expressed recombinant plasmids

The co-expression recombinant plasmid pRSFDuet-LG-23/T438S _17 beta-HSDcl is transformed into E.coli BL21 competent cells, added with a culture medium for 1h to recover, then evenly spread on a solid LB plate containing 50 ul/mg kanamycin, and cultured at 37 ℃ overnight. The subsequent bacteria culture method and reaction conditions are the same as the functional verification part of example 1, the whole-cell catalysis reaction process and detection result of Escherichia coli co-expressing P450BM 3-LG-23/T438S and 17 beta-HSDcl for converting 4,9 into 11 alpha-hydroxymethyl dienolone are shown in figure 2, wherein figure 2-a is a reaction flow chart, in the figure, 1 represents 4,9, 1a represents 11 alpha-hydroxy 4,9, 1b represents methyl dienolone, and 2 represents 11 alpha-hydroxymethyl dienolone; FIG. 2-b shows the HPLC analysis results at 0min, 5min, 20min and 120 min; fig. 2-c is the relative proportion of each steroid over reaction time.

The results show that when the reaction time reaches 2 hours, the substrates 4 and 9 are completely converted into 11 alpha-hydroxymethyl dienolone, the product proportion reaches 90 percent, and the intermediate products 11 alpha-hydroxy 4,9 and methyl dienolone are basically free of residues.

3. Preparation of glucose dehydrogenase GDH crude enzyme freeze-dried powder

Transferring the GDH enzyme gene from Bacillus megaterium (Priesia megaterium) into E.coli BL21 by a conventional method in the field, and inducing expression by IPTG at the induction temperature of 25 ℃ for 14-16 h. The culture solution is centrifuged at 4000rpm and 4 ℃ for 10min, the cells are collected, the cells are washed once by 100mM potassium phosphate buffer solution (pH8.0), then the cells are suspended in a 50mL bacteria shaking tube by 100mM potassium phosphate buffer solution (pH8.0) with a certain volume, the cells are placed in an ice-water mixture for ultrasonic disruption (power 350W, working time 2s, interval time 4s and 15min), after the solution becomes clear and transparent, the cells are centrifuged at 9000rpm and 4 ℃ for 30min for separation to obtain disrupted supernatant and cell sediment, the small cells are filled in a culture dish and are lyophilized to be powder by a vacuum freeze dryer, and the cells can be stored for a long time at-20 ℃. If no large amount of long-term needs exist, the GDH bacteria-breaking supernatant can be directly used for experiment, and the bacteria-breaking supernatant can be stored at-80 ℃ for short-term use.

And (3) carrying out enzyme activity determination on the obtained GDH crude enzyme freeze-dried powder, and specifically operating as follows: 1mg of GDH crude enzyme lyophilized powder was dissolved in 940uL of 100mM potassium phosphate buffer (pH8.0) in a cuvette, and 20uL of 50mM NADP was added⁺Scanning with spectrophotometer at 340nm for 1min as blank control; immediately after adding 20uL of 50% glucose to the cuvette and rapidly mixing, time scanning was performed, and the amount of a substance (umol) of NADPH generated within 1min was calculated from the NADPH concentration and the absorbance at 340 nm. The experiment was repeated three times. And calculating the enzyme activity of the GDH, and if the amount of the generated NADPH substance is X umol, the enzyme activity of the GDH freeze-dried powder is X U/mg.

EXAMPLE 3 Synthesis of trenbolone acetate

In this embodiment, trenbolone acetate is generated by combining biological enzyme catalysis and chemical catalysis, wherein the biological enzyme catalyzes the 4,9 product to generate 11 α -hydroxymethyl dienolone by one-step method, the trenbolone is generated by chemical dehydration, and the trenbolone acetate is obtained by esterification reaction of trenbolone and acetic anhydride, wherein the reaction flow chart is shown in fig. 3.

The specific experimental process comprises the following steps:

1. hydroxylation/reduction reaction

The whole cells of E.coli co-expressing P450BM 3-LG-23/T438S and 17 β -HSDcl cultured in example 2 were resuspended (OD 8.0 in 4L 100mM potassium phosphate buffer)₆₀₀20-30) in a 10L reaction tank, and stirring at 25 ℃ and 400 rpm. 1g of 4, 9-substance was dissolved in 40mL of DMF, and the solution was put into a reaction vessel, to which 0.7g of NADP was added⁺After adding 100g of glucose and 2000U of GDH crude enzyme, stirring and reacting for 2-3 hours at 25 ℃ until the TLC analysis raw material reaction is complete. Then adding 4L ethyl acetate to extract the reaction solution for three times, combining all ethyl acetate extract, dehydrating with anhydrous sodium sulfate, filtering, concentrating under reduced pressure until no distillate exists, obtaining 11 alpha-hydroxymethyl diketene (II) crude product, wherein LC/MS analysis spectrum is shown in figure 3.

2. Dehydration reaction

Dissolving the crude product of the 11 alpha-hydroxymethyl dienolone (II) in 25mL of trichloromethane, adding 0.66g of p-toluenesulfonic acid monohydrate, stirring at room temperature for reaction until TLC analysis of raw materials completely reacts, and adding 10mL of saturated sodium carbonate solution to stop the reaction. The aqueous phase was then extracted twice with 10mL of chloroform; combining all trichloromethane, dehydrating with anhydrous sodium sulfate, filtering, concentrating under reduced pressure to small volume, separating and purifying the target product by silica gel column chromatography, and concentrating the collected chromatographic solution under reduced pressure to dryness to obtain trenbolone (III) 0.729g with a mass yield of 72.9% (calculated on 4 and 9 substances) and an HPLC content of 98.3%.

3. Esterification reaction

Dissolving 1g of trenbolone (III) in 15mL of dichloromethane, adding 25mg of Dimethylaminopyridine (DMAP) and 1.2g of acetic anhydride, controlling the temperature to be 20-25 ℃, stirring for reacting for 2 hours, analyzing by TLC that the raw materials are completely reacted, washing by using 10mL of 5% sodium carbonate aqueous solution, and extracting the water phase twice by using 10mL of dichloromethane; mixing all dichloromethane, dehydrating with anhydrous sodium sulfate, filtering, concentrating under reduced pressure until no distillate exists, adding isopropyl ether, adding 2mL isopropyl ether, cooling, crystallizing, and centrifuging. Drying to obtain 1.066g of trenbolone acetate with the mass yield of 106.6%, the HPLC content of more than or equal to 99.0% and the total mass yield of 77.7% (calculated by 4 and 9 substances).

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Sequence listing

<110> university of Hubei

<120> cytochrome P450BM3 mutant and application thereof in synthesis of trenbolone acetate

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Ala Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn

1 5 10 15

Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile

20 25 30

Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Trp Val

35 40 45

Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu

50 55 60

Ser Arg Phe Asp Lys Asn Leu Trp Gln Ala Leu Lys Tyr Leu Arg Asp

65 70 75 80

Ile Leu Gly Asp Gly Leu Gly Ser Ser Trp Thr His Glu Lys Asn Trp

85 90 95

Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met

100 105 110

Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln

115 120 125

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

130 135 140

Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr

145 150 155 160

Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser

165 170 175

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

180 185 190

Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp

195 200 205

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

210 215 220

Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly

225 230 235 240

Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr

245 250 255

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

260 265 270

Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln

275 280 285

Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser

290 295 300

Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu

305 310 315 320

Ala Leu Arg Leu Trp Pro Thr Gly Pro Trp Phe Ser Leu Tyr Ala Lys

325 330 335

Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu

340 345 350

Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly

355 360 365

Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala

370 375 380

Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys

385 390 395 400

Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met

405 410 415

Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp

420 425 430

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

435 440 445

Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu

450 455 460

Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala His Asn Thr

465 470 475 480

Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr

485 490 495

Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln

500 505 510

Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala

515 520 525

Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala

530 535 540

Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val Lys

545 550 555 560

Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr

565 570 575

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

580 585 590

Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp

595 600 605

Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val

610 615 620

Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser

625 630 635 640

Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala

645 650 655

Lys Met His Gly Ala Phe Ser Thr Asn Val Val Ala Ser Lys Glu Leu

660 665 670

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

675 680 685

Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro

690 695 700

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

705 710 715 720

Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala

725 730 735

His Leu Pro Leu Ala Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr

740 745 750

Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala

755 760 765

Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu

770 775 780

Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met

785 790 795 800

Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys Phe Ser Glu

805 810 815

Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro Arg Tyr Tyr Ser Ile Ser

820 825 830

Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val

835 840 845

Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala

850 855 860

Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe

865 870 875 880

Ile Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp Pro Glu Thr

885 890 895

Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly

900 905 910

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

915 920 925

Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu

930 935 940

Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser Glu Gly Ile Ile Thr Leu

945 950 955 960

His Thr Ala Phe Ser Arg Met Pro Asn Gln Pro Lys Thr Tyr Val Gln

965 970 975

His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln

980 985 990

Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Ala

995 1000 1005

Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val His Gln Val Ser

1010 1015 1020

Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg

1025 1030 1035 1040

Tyr Ala Lys Asp Val Trp Ala Gly

1045

<210> 2

<211> 1048

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Ala Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn

1 5 10 15

Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile

20 25 30

Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Trp Val

35 40 45

Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu

50 55 60

Ser Arg Phe Asp Lys Asn Leu Trp Gln Ala Leu Lys Tyr Leu Arg Asp

65 70 75 80

Ile Leu Gly Asp Gly Leu Gly Ser Ser Trp Thr His Glu Lys Asn Trp

85 90 95

Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met

100 105 110

Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln

115 120 125

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

130 135 140

Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr

145 150 155 160

Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser

165 170 175

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

180 185 190

Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp

195 200 205

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

210 215 220

Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly

225 230 235 240

Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr

245 250 255

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

260 265 270

Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln

275 280 285

Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser

290 295 300

Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu

305 310 315 320

Ala Leu Arg Leu Trp Pro Thr Gly Pro Trp Phe Ser Leu Tyr Ala Lys

325 330 335

Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu

340 345 350

Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly

355 360 365

Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala

370 375 380

Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys

385 390 395 400

Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met

405 410 415

Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp

420 425 430

Ile Lys Glu Thr Leu Ala Leu Lys Pro Glu Gly Phe Val Val Lys Ala

435 440 445

Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu

450 455 460

Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala His Asn Thr

465 470 475 480

Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr

485 490 495

Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln

500 505 510

Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala

515 520 525

Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala

530 535 540

Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val Lys

545 550 555 560

Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr

565 570 575

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

580 585 590

Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp

595 600 605

Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val

610 615 620

Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser

625 630 635 640

Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala

645 650 655

Lys Met His Gly Ala Phe Ser Thr Asn Val Val Ala Ser Lys Glu Leu

660 665 670

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

675 680 685

Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro

690 695 700

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

705 710 715 720

Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala

725 730 735

His Leu Pro Leu Ala Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr

740 745 750

Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala

755 760 765

Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu

770 775 780

Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met

785 790 795 800

Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys Phe Ser Glu

805 810 815

Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro Arg Tyr Tyr Ser Ile Ser

820 825 830

Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val

835 840 845

Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala

850 855 860

Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe

865 870 875 880

Ile Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp Pro Glu Thr

885 890 895

Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly

900 905 910

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

915 920 925

Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu

930 935 940

Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser Glu Gly Ile Ile Thr Leu

945 950 955 960

His Thr Ala Phe Ser Arg Met Pro Asn Gln Pro Lys Thr Tyr Val Gln

965 970 975

His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln

980 985 990

Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Ala

995 1000 1005

Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val His Gln Val Ser

1010 1015 1020

Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg

1025 1030 1035 1040

Tyr Ala Lys Asp Val Trp Ala Gly

1045

<210> 3

<211> 1048

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Ala Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys Asn

1 5 10 15

Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys Ile

20 25 30

Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Trp Val

35 40 45

Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp Glu

50 55 60

Ser Arg Phe Asp Lys Asn Leu Trp Gln Ala Leu Lys Tyr Leu Arg Asp

65 70 75 80

Ile Leu Gly Asp Gly Leu Gly Ser Ser Trp Thr His Glu Lys Asn Trp

85 90 95

Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala Met

100 105 110

Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val Gln

115 120 125

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

130 135 140

Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn Tyr

145 150 155 160

Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr Ser

165 170 175

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

180 185 190

Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu Asp

195 200 205

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

210 215 220

Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn Gly

225 230 235 240

Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg Tyr

245 250 255

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

260 265 270

Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu Gln

275 280 285

Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro Ser

290 295 300

Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn Glu

305 310 315 320

Ala Leu Arg Leu Trp Pro Thr Gly Pro Trp Phe Ser Leu Tyr Ala Lys

325 330 335

Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu

340 345 350

Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp Gly

355 360 365

Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser Ala

370 375 380

Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala Cys

385 390 395 400

Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met

405 410 415

Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp

420 425 430

Ile Lys Glu Thr Leu Gly Leu Lys Pro Glu Gly Phe Val Val Lys Ala

435 440 445

Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr Glu

450 455 460

Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala His Asn Thr

465 470 475 480

Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr

485 490 495

Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln

500 505 510

Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala

515 520 525

Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala

530 535 540

Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val Lys

545 550 555 560

Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr

565 570 575

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

580 585 590

Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp Asp

595 600 605

Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Val

610 615 620

Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys Ser

625 630 635 640

Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala

645 650 655

Lys Met His Gly Ala Phe Ser Thr Asn Val Val Ala Ser Lys Glu Leu

660 665 670

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

675 680 685

Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro

690 695 700

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

705 710 715 720

Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala

725 730 735

His Leu Pro Leu Ala Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr

740 745 750

Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala

755 760 765

Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu Leu

770 775 780

Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met

785 790 795 800

Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys Phe Ser Glu

805 810 815

Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro Arg Tyr Tyr Ser Ile Ser

820 825 830

Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val

835 840 845

Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala

850 855 860

Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe

865 870 875 880

Ile Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp Pro Glu Thr

885 890 895

Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly

900 905 910

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

915 920 925

Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu

930 935 940

Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser Glu Gly Ile Ile Thr Leu

945 950 955 960

His Thr Ala Phe Ser Arg Met Pro Asn Gln Pro Lys Thr Tyr Val Gln

965 970 975

His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln

980 985 990

Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro Ala

995 1000 1005

Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val His Gln Val Ser

1010 1015 1020

Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly Arg

1025 1030 1035 1040

Tyr Ala Lys Asp Val Trp Ala Gly

1045

<210> 4

<211> 3147

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gcaattaaag aaatgcctca gccaaaaacg tttggagagc ttaaaaattt accgttatta 60

aacacagata aaccggttca agctttgatg aaaattgcgg atgaattagg agaaatcttt 120

aaattcgagg cgcctggttg ggtaacgcgc tacttatcaa gtcagcgtct aattaaagaa 180

gcatgcgatg aatcacgctt tgataaaaac ttatggcaag cgcttaaata cctaagagac 240

atcctaggag acgggttagg atcaagctgg acgcatgaaa aaaattggaa aaaagcgcat 300

aatatcttac ttccaagctt cagtcagcag gcaatgaaag gctatcatgc gatgatggtc 360

gatatcgccg tgcagcttgt tcaaaagtgg gagcgtctaa atgcagatga gcatattgaa 420

gtaccggaag acatgacacg tttaacgctt gatacaattg gtctttgcgg ctttaactat 480

cgctttaaca gcttttaccg agatcagcct catccattta ttacaagtac ggtccgtgca 540

ctggatgaag cacaaaacaa gcaacagcga gcaaatccag acgacccagc ttatgatgaa 600

aacaagcgcc agttccaaga agatacaaag gtgatgaacg acctagtaga taaaattatt 660

gcagatcgca aagcaagcgg tgaacaaagc gatgatttat taacgcatat gctaaacgga 720

aaagatccag aaacgggtga gccgcttgat gacgagaaca ttcgctatca aattattaca 780

ttcttaattg cgggacacga aacaacaagt ggtcttttat catttgcgct gtatttctta 840

gtgaaaaatc cacatgtatt acaaaaagca gcagaagaag cagcacgagt tctagtagat 900

cctgttccaa gctacaaaca agtcaaacag cttaaatatg tcggcatggt cttaaacgaa 960

gcgctgcgct tatggccaac tggtccttgg ttttccctat atgcaaaaga agatacggtg 1020

cttggaggag aatatccttt agaaaaaggc gacgaactaa tggttctgat tcctcagctt 1080

caccgtgata aaacaatttg gggagacgat gtggaagagt tccgtccaga gcgttttgaa 1140

aatccaagtg cgattccgca gcatgcgttt aaaccgtttg gaaacggtca gcgtgcgtgt 1200

atcggtcagc agttcgctct tcatgaagca acgctggtac ttggtatgat gctaaaacac 1260

tttgactttg aagatcatac aaactacgag ctggatatta aagaaacttt atctttaaaa 1320

cctgaaggct ttgtggtaaa agcaaaatcg aaaaaaattc cgcttggcgg tattccttca 1380

cctagcactg aacagtctgc taaaaaagta cgcaaaaagg cagaaaacgc tcataatacg 1440

ccgctgcttg tgctatacgg ttcaaatatg ggaacagctg aaggaacggc gcgtgattta 1500

gcagatattg caatgagcaa aggatttgca ccgcaggtcg caacgcttga ttcacacgcc 1560

ggaaatcttc cgcgcgaagg agctgtatta attgtaacgg cgtcttataa cggtcatccg 1620

cctgataacg caaagcaatt tgtcgactgg ttagaccaag cgtctgctga tgaagtaaaa 1680

ggcgttcgct actccgtatt tggatgcggc gataaaaact gggctactac gtatcaaaaa 1740

gtgcctgctt ttatcgatga aacgcttgcc gctaaagggg cagaaaacat cgctgaccgc 1800

ggtgaagcag atgcaagcga cgactttgaa ggcacatatg aagaatggcg tgaacatatg 1860

tggagtgacg tagcagccta ctttaacctc gacattgaaa acagtgaaga taataaatct 1920

actctttcac ttcaatttgt cgacagcgcc gcggatatgc cgcttgcgaa aatgcacggt 1980

gcgttttcaa cgaacgtcgt agcaagcaaa gaacttcaac agccaggcag tgcacgaagc 2040

acgcgacatc ttgaaattga acttccaaaa gaagcttctt atcaagaagg agatcattta 2100

ggtgttattc ctcgcaacta tgaaggaata gtaaaccgtg taacagcaag gttcggccta 2160

gatgcatcac agcaaatccg tctggaagca gaagaagaaa aattagctca tttgccactc 2220

gctaaaacag tatccgtaga agagcttctg caatacgtgg agcttcaaga tcctgttacg 2280

cgcacgcagc ttcgcgcaat ggctgctaaa acggtctgcc cgccgcataa agtagagctt 2340

gaagccttgc ttgaaaagca agcctacaaa gaacaagtgc tggcaaaacg tttaacaatg 2400

cttgaactgc ttgaaaaata cccggcgtgt gaaatgaaat tcagcgaatt tatcgccctt 2460

ctgccaagca tacgcccgcg ctattactcg atttcttcat cacctcgtgt cgatgaaaaa 2520

caagcaagca tcacggtcag cgttgtctca ggagaagcgt ggagcggata tggagaatat 2580

aaaggaattg cgtcgaacta tcttgccgag ctgcaagaag gagatacgat tacgtgcttt 2640

atttccacac cgcagtcaga atttacgctg ccaaaagacc ctgaaacgcc gcttatcatg 2700

gtcggaccgg gaacaggcgt cgcgccgttt agaggctttg tgcaggcgcg caaacagcta 2760

aaagaacaag gacagtcact tggagaagca catttatact tcggctgccg ttcacctcat 2820

gaagactatc tgtatcaaga agagcttgaa aacgcccaaa gcgaaggcat cattacgctt 2880

cataccgctt tttctcgcat gccaaatcag ccgaaaacat acgttcagca cgtaatggaa 2940

caagacggca agaaattgat tgaacttctt gatcaaggag cgcacttcta tatttgcgga 3000

gacggaagcc aaatggcacc tgccgttgaa gcaacgctta tgaaaagcta tgctgacgtt 3060

caccaagtga gtgaagcaga cgctcgctta tggctgcagc agctagaaga aaaaggccga 3120

tacgcaaaag acgtgtgggc tgggtaa 3147

<210> 5

<211> 3147

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5