Ethylenic bond reductase mutant with improved activity and stereoselectivity as well as encoding gene and application thereof

文档序号：81180 发布日期：2021-10-08 浏览：37次中文

阅读说明：本技术 一种活性和立体选择性提高的烯键还原酶突变体及其编码基因以及应用 (Ethylenic bond reductase mutant with improved activity and stereoselectivity as well as encoding gene and application thereof ) 是由林金萍魏东芝张宝琪杜涵于 2021-06-17 设计创作，主要内容包括：本发明公开了一种活性和立体选择性提高的烯键还原酶突变体及其编码基因以及应用,该烯键还原酶突变体包括以SEQ ID NO.1所示野生型的烯键还原酶OYE2p为模板,第84位氨基酸残基发生突变形成的烯键还原酶突变体Y84A,Y84V,Y84L,Y84I,Y84T,Y84C,氨基酸序列如SEQ ID NO.3～8所示。根据本发明提供的烯键还原酶的突变体可高活性、高立体选择性催化柠檬醛制备(R)-香茅醛,具有经济环保、手性选择性高的特点,为(R)-香茅醛的工业化生产提供一个潜在的生物催化剂。(The invention discloses an olefinic bond reductase mutant with improved activity and stereoselectivity, a coding gene and application thereof, wherein the olefinic bond reductase mutant comprises an olefinic bond reductase mutant Y84A, Y84V, Y84L, Y84I, Y84T and Y84C which are formed by mutating 84 th amino acid residue by using a wild type olefinic bond reductase OYE2p shown in SEQ ID NO.1 as a template, and the amino acid sequence is shown in SEQ ID NO. 3-8. The mutant of the olefinic bond reductase provided by the invention can catalyze citral to prepare (R) -citronellal with high activity and high stereoselectivity, has the characteristics of economy, environmental protection and high chiral selectivity, and provides a potential biocatalyst for industrial production of (R) -citronellal.)

1. An olefinic bond reductase mutant with improved activity and stereoselectivity, which is characterized by comprising an olefinic bond reductase mutant formed by taking a wild type olefinic bond reductase OYE2p shown in SEQ ID NO.1 as a template and carrying out the following mutations on amino acid residues at position 84:

the tyrosine Y at the 84 th position is mutated into an olefinic bond reductase mutant Y84A of alanine A, and the amino acid sequence is shown as SEQ ID NO. 3;

an olefinic bond reductase mutant Y84V with the mutation of tyrosine Y at the 84 th position into valine V and the amino acid sequence shown as SEQ ID NO. 4;

an olefinic bond reductase mutant Y84L with the 84 th tyrosine Y mutated into leucine L and the amino acid sequence shown as SEQ ID NO. 5;

tyrosine Y at the 84 th position is mutated into an olefinic bond reductase mutant Y84I of isoleucine I, and the amino acid sequence is shown as SEQ ID NO. 6;

an olefinic bond reductase mutant Y84T with the 84 th tyrosine Y mutated into threonine T and the amino acid sequence shown as SEQ ID NO. 7;

tyrosine Y at the 84 th position is mutated into an olefinic bond reductase mutant Y84C of cysteine C, and the amino acid sequence is shown as SEQ ID NO. 8.

2. A gene encoding an olefinic bond reductase mutant, wherein the gene encodes the amino acid sequence of any one of the olefinic bond reductase mutants of claim 1.

3. A recombinant genetically engineered bacterium comprising a gene encoding the ethylenic reductase mutant of claim 2.

4. The recombinant genetically engineered bacterium of claim 3, wherein the recombinant genetically engineered bacterium is a host bacterium of Escherichia coli.

5. Use of the mutant of an ethylenic reductase according to claim 1 for catalyzing citral to produce (R) -citronellal.

6. The use of claim 5, wherein the use comprises the preparation of (R) -citronellal from citral catalyzed by a recombinant genetically engineered bacterium containing a gene encoding an olefinic bond reductase mutant.

7. The application of claim 6, wherein the application comprises: the method comprises the steps of taking thalli obtained by centrifuging recombinant genetic engineering bacteria containing olefinic bond reductase mutant coding genes after fermentation culture, thalli immobilized cells, enzyme extracted after ultrasonic disruption of the thalli, immobilized enzyme or pure enzyme as a catalyst, taking citral as a substrate, taking a buffer solution with the pH of 7-9 as a reaction medium, reacting at the temperature of 20-40 ℃ and under the condition of 300R/min, and after the reaction is finished, separating and purifying a reaction liquid to obtain (R) -citronellal.

8. The use according to claim 7, wherein the substrate concentration in the reaction system is 20 to 200mM, the glucose concentration is 30 to 220mM, the amount of the ethylenic reductase to be used is 1 to 5U/mL in terms of the enzyme activity, and the amount of the glucose dehydrogenase to be used is 1.5 to 7.5U/mL in terms of the enzyme activity.

9. Use according to claim 8, wherein the reaction is carried out by adding Na, depending on the pH change₂CO₃The solution was adjusted to pH 8.5.

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to an olefinic bond reductase mutant with improved activity and stereoselectivity, and a coding gene and application thereof.

Background

The (R) -Citronellal ((R) - (+) -Citronellal) is chemically (3R) -3, 7-dimethyl-6-octenal, has a chemical structural formula shown as (I), is an important spice and a medical intermediate, can be used for blending flavors of foods, daily necessities and the like, and can also be used as a raw material for synthesizing L-menthol and vitamin E. At present, (R) -citronellal is mainly obtained by separating and extracting natural essential oil, and can also be prepared by catalytic hydrogenation of citral through a metal catalyst.

Compared with other methods, the method for synthesizing (R) -citronellal by catalyzing citral through the olefinic bond reductase and carrying out asymmetric reduction has the advantages of environmental friendliness, mild reaction conditions, high product stereoselectivity and the like. The currently reported olefinic bond reductases capable of catalyzing citral to generate (R) -citronellal are few, and the catalytic activity is low. Stewart et al obtained an olefinic bond reductase OYE2.6 from Pichia stipitis which catalyzes the formation of (R) -citronellal from 150mM E-citral within 5.75h with a product ee value of 98% [ Chemical Communications,2010,46(45):8558- ] 8560; however, the substrate E-citral is obtained by separating citral (mixture of E, Z-citral). Hauer et al have engineered an olefinic bond reductase NCR derived from Zymomonas mobilis by means of protein engineering, and finally the enantioselectivity of the mutant strain W66A to E-citral is reversed from 99% (S) to 46% (R), but the enantioselectivity to Z-citral is only 88% (S), so that the requirement for producing (R) -citronellal is difficult to meet. Therefore, the development of an ethylenic reductase capable of catalyzing citral to (R) -citronellal with high activity and high selectivity has important industrial application value.

Disclosure of Invention

The invention aims to provide an olefinic bond reductase mutant with improved activity and stereoselectivity, a coding gene and application thereof, so as to solve the problems of low stereospecific activity and low stereoselectivity in the existing technology for synthesizing (R) -citronellal by catalytic catalysis of olefinic bond reductase.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to a first aspect of the present invention, there is provided an olefinic bond reductase mutant with improved activity and stereoselectivity, comprising an olefinic bond reductase mutant obtained by mutating amino acid 84 as follows, wherein the olefinic bond reductase mutant is OYE2p as a template, and has an amino acid sequence as shown in SEQ ID No.1 and a nucleotide sequence as shown in SEQ ID No. 2: an ethylenic reductase mutant Y84A (SEQ ID NO.3) in which tyrosine Y at position 84 is mutated to alanine A; an olefinic bond reductase mutant Y84V (SEQ ID NO.4) in which tyrosine Y at position 84 is mutated to valine V; an ethylenic reductase mutant Y84L (SEQ ID NO.5) in which tyrosine Y at position 84 is mutated to leucine L; an olefinic bond reductase mutant Y84I (SEQ ID NO.6) in which the tyrosine Y at position 84 is mutated into isoleucine I; an ethylenic reductase mutant Y84T (SEQ ID NO.7) in which tyrosine Y at position 84 is mutated to threonine T; an ethylenic reductase mutant Y84C (SEQ ID NO.8) in which tyrosine Y at position 84 is mutated to cysteine C.

According to the invention, a deposited olefinic bond reductase OYE2p (derived from Saccharomyces cerevisiae YJM1341, see document Bioresource. bioprocess. (2018)5:9) is constructed at a laboratory early stage and used as a template, the amino acid sequence of the olefinic bond reductase is shown as SEQ ID No.1, the nucleotide sequence is shown as SEQ ID No.2, and the olefinic bond reductase mutant with obviously improved catalytic activity, stereoselectivity and the like is obtained by carrying out site-directed mutagenesis on the olefinic bond reductase.

According to a second aspect of the present invention, there is provided a gene encoding an olefinic bond reductase mutant having improved enzymatic activity and stereoselectivity as described above, the gene encoding an amino acid sequence shown in the olefinic bond reductase mutant as described above.

Particularly preferably, the invention provides an olefinic bond reductase mutant Y84V with improved enzyme activity and stereoselectivity, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 9.

According to the third aspect of the invention, the recombinant gene engineering bacteria containing the coding gene of the olefinic bond reductase mutant with improved enzyme activity and stereoselectivity are also provided. The host cell may be any of the various conventional host cells in the art, and preferably, the host cell is E.coli BL21(DE 3).

According to the fourth aspect of the invention, the application of the ethylenic reductase mutant in catalyzing citral to prepare (R) -citronellal is also provided.

The application takes engineering bacteria containing olefinic bond reductase mutant coding genes, thalli obtained by centrifugation after fermentation culture, thalli immobilized cells, enzyme extracted after ultrasonic disruption of the thalli, immobilized enzyme or pure enzyme as a catalyst, citral as a substrate, a buffer solution with the pH value of 7-9 as a reaction medium, and the reaction is carried out under the conditions of 20-40 ℃, 150-300rpm, and after the reaction is finished, the reaction liquid is separated and purified to obtain (R) -citronellal.

It should be understood that the olefinic bond reductase mutant of the present invention can be used in the form of whole cells of the engineered bacterium, crude enzyme without purification, or partially purified or completely purified enzyme. The mutant ethylenic reductase of the present invention may also be prepared into biocatalysts in the form of immobilized enzymes or immobilized cells using immobilization techniques known in the art.

Preferably, the concentration of the substrate is 50-200mM, the concentration of the glucose is 55-220mM, the dosage of the crude enzyme powder of the ethylenic bond reductase is 2U/mL by the enzyme activity, and the dosage of the crude enzyme powder of the glucose dehydrogenase is 3U/mL by the enzyme activity.

Preferably, the reaction medium is PBS buffer solution with pH value of 8.5, and the temperature of the catalytic reaction is 30 ℃.

Preferably, the crude enzyme powder of the olefinic bond reductase is a lyophilized olefinic bond reductase mutant Y84V.

The medium used for recombinant expression of the transformant may be a medium which allows the transformant to grow and produce the ethylenic reductase of the present invention in the art, and is preferably an LB medium: 10g/L of peptone, 10g/L of sodium chloride, 5g/L of yeast extract and 7.0 of pH.

The culture method and culture conditions are not particularly limited as long as the transformant can grow and express the ethylenic reductase. The method specifically comprises the following steps:

(1) plate culture: streaking the recombinant engineering bacteria related to the invention on an LB solid plate culture medium containing screening antibiotics, and culturing overnight at 37 ℃;

(2) seed culture: picking a single colony on the plate obtained in the step (1) on an ultra-clean bench, inoculating the single colony into an LB liquid culture medium containing screening antibiotics, and culturing for 8h at 37 ℃;

(3) and (3) induction culture: inoculating the seed culture solution obtained in the step (2) into an LB liquid culture medium containing screening antibiotics on a super clean bench, culturing at 37 ℃ until OD of a bacterial solution is obtained₆₀₀When the value reaches 0.6, adding IPTG with the final concentration of 0.2mM, and carrying out induced culture at 20 ℃ for 12 h;

(4) and (3) collecting thalli: centrifuging the bacterial liquid obtained by the induced culture in the step (3) for 10min under the condition that the rotating speed is 8000rpm, and separating to obtain thallus sediment; washing the obtained thallus with normal saline for 2 times;

(5) protein purification: and (3) dissolving the thalli obtained in the step (4) in 20mM PBS buffer solution (pH7.4) to obtain cell suspension, performing ultrasonic disruption, centrifuging for 30min under the condition that the rotating speed is 10000rpm, separating to obtain a supernatant crude enzyme solution, and purifying by a nickel column to obtain a completely purified olefinic bond reductase mutant, namely the catalyst.

Preferably, the concentration of the cell suspension in the step (5) is 50 g/L.

Preferably, the ultrasonic crushing power in the step (5) is 200-300W, 5s of ultrasonic treatment, 5s of pause and 99 times of accumulation.

According to the present invention, it was found that position 84 of the wild-type ethylenic reductase plays a key role in both the enhancement of activity and stereoselectivity. Particularly preferably, the olefinic bond reductase mutant Y84V provided by the invention can catalyze citral to prepare (R) -citronellal with high activity and high stereoselectivity, has the characteristics of economy, environmental protection and high chiral selectivity, and provides a potential biocatalyst for industrial production of (R) -citronellal.

Compared with the prior art, the invention has the following beneficial effects:

(1) the olefinic bond reductase mutant Y84V provided by the invention can catalyze high-concentration citral (200mM) to generate (R) -citronellal, the conversion rate is more than 95%, and the ee value of the product (R) -citronellal is more than 95%;

(2) the invention improves the activity and stereoselectivity of the olefinic bond reductase through protein engineering, and has good application prospect in the industrial production of (R) -citronellal.

Drawings

FIG. 1 is a SDS-PAGE protein gel electrophoresis of wild-type ethylenic reductase OYE2P and its mutant Y84V, in which M is protein standard molecular weight, T is whole cell fraction, S is cell disruption supernatant, P is cell disruption precipitate, and E is pure enzyme;

FIG. 2 is a graph of the course of the reaction of wild-type ethylenic reductase OYE2p in catalyzing citral;

FIG. 3 is a graph of the course of the reaction of the ethylenic reductase OYE2p mutant Y84V catalyzing citral.

Detailed Description

The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention. The techniques used in the examples are conventional in the art, as specifically described.

The parent olefinic bond reductase adopted in the specific embodiment is an olefinic bond reductase OYE2p constructed in the early stage of the subject group, the amino acid sequence of the parent olefinic bond reductase is shown as SEQ ID NO.1, and the nucleotide sequence of the parent olefinic bond reductase is shown as SEQ ID NO. 2.

EXAMPLE 1 construction of recombinant E.coli containing mutants

In order to perform site-directed mutagenesis on tyrosine (Tyr) at the 84 th site in the amino acid sequence of parent olefinic bond reductase, mutation sites Y84A, Y84V, Y84L, Y84I, Y84T and Y84C are designed on primers, and plasmids with mutated base sequences are amplified by PCR by using a recombinant plasmid pET21a-OYE2p containing a target gene fragment as a template, and the sequences of the primers are shown in Table 1.

Table 1: primer design sheet

The PCR amplification system was (50. mu.L): template DNA 1-5ng, PrimeSTAR Max Premix (2X) 25. mu.L, mutation primer upstream and downstream each 1.5. mu.L, with sterile distilled water to make up to 50. mu.L.

PCR reaction parameters: (1) pre-denaturation at 98 ℃ for 2 min; (2) denaturation at 95 ℃ for 10 seconds; (3) annealing at 55 ℃ for 5 seconds; (4) extension at 72 ℃ for 2 min, and 30 cycles of steps (2) - (4); (5) extension was completed at 72 ℃ for 5 minutes, and storage was carried out at 16 ℃.

After observing a target band of the PCR product through 1.0% agarose gel electrophoresis, purifying the residual PCR product to obtain a purified linearized plasmid, and taking 50ng of the purified linearized plasmid for seamless cloning.

The seamless cloning system was (20 μ L): linearized plasmid 50ng, Seamless Cloning Mix (2X) 5. mu.L, supplemented to 10. mu.L with sterile distilled water.

The seamless cloning reaction was carried out in a water bath at 50 ℃ for 15 minutes.

The seamless clone product 5. mu.L was taken and transformed into E.coli BL21(DE3) competent cells by heat shock, and then recovered and plated on LB plate containing ampicillin to culture overnight.

Then 5-10 clones are picked to LB culture medium, cultured for 8h at 37 ℃, and then taken bacterial liquid is subjected to sequencing validation to obtain the olefinic bond reductase mutant recombinant engineering bacteria E.coli BL21(DE3)/pET21a-OYE2p/Y84A, E.coli BL21(DE3)/pET21a-OYE2p/Y84V, E.coli BL V (DE V)/pET 21V-OYE 2V/Y84V and E.coli BL V (DE V)/pET 21V-OYE 2V/Y84, and the amino acid sequence of the olefinic bond reductase SEQ ID NO.3 is shown as SEQ ID reductase sequence NO. 3.

Example 2 expression and purification of wild type OYE2p and each mutant

Step 1: the olefinic bond reductase OYE2p obtained in example 1 and the mutant recombinant engineering bacteria were streaked onto LB solid plate medium containing the selected antibiotic, cultured overnight at 37 ℃, single colonies on a single plate were picked up, inoculated into LB liquid medium containing the selected antibiotic, and cultured for 8 hours at 37 ℃. Inoculating the seed culture solution into 200mL LB liquid culture medium containing screening antibiotics, culturing at 37 ℃, adding IPTG with final concentration of 0.2mM when OD600 value of the bacterial solution reaches 0.6, and inducing and culturing at 20 ℃ for 12 h.

Step 2: wild type OYE2p and 6 mutants thereof were purified by immobilized metal ion affinity method using HisTrap from GE as nickel column^TMHP (5 mL). Centrifuging the obtained bacterial liquid for 10min at 8000rpm, separating to obtain thallus precipitate, dissolving the thallus in 20mM PBS buffer (pH7.4) to obtain cell suspension, ultrasonically crushing, centrifuging at 10000rpm for 30min, collecting supernatant, and filtering with 0.22 μm water system filter membrane to obtain crude enzyme liquid.

And step 3: and (3) after the nickel column is balanced by the balance buffer solution, sampling the crude enzyme solution obtained in the step (2), removing foreign proteins by using a low-concentration elution buffer solution, eluting and collecting the target protein by using a high-concentration elution buffer solution, and dialyzing to obtain the pure enzyme. Protein concentration was quantified using the Bradford protein concentration assay kit. The protein purity was checked by SDS-PAGE and the results are shown in FIG. 1, which shows that both wild type and mutant purified to obtain a single protein band.

Example 3 Activity and stereoselectivity of wild type OYE2p and the mutants on citral

The OYE2p obtained in the step 2 of the example 2 and the crude enzyme solution of each mutant expression strain are frozen and dried to prepare freeze-dried crude enzyme powder.

The activity of citral and the ee value of the product were determined by catalyzing the reduction of citral with wild type OYE2p and each mutant in the presence of a uniform amount of enzyme.

The enzyme catalytic reduction reaction system comprises: 100mM phosphate buffer (pH8.5), 15g/L of lyophilized wild type OYE2p and each mutant crude enzyme powder, 5g/L of lyophilized glucose dehydrogenase crude enzyme powder, 20mM citral, 0.2mM NAD⁺And 100mM glucose. Sequentially adding the substances into a centrifugal tube, placing the centrifugal tube at 200rpm and 30 ℃ for oscillation reaction for 6h, adding equal volume of ethyl acetate to extract twice after the reaction is finished, and combining organic phases for gas phaseAnd (6) detecting.

The results show that the catalytic activity of mutants Y84A, Y84V, Y84L, Y84I, Y84T and Y84C is increased by 25.2%, 40.0%, 25.5%, 34.5%, 24.7% and 33.75% respectively compared with the wild type. And the products of wild type OYE2p and each mutant catalyzing citral are mainly (R) -citronellal, wherein the stereoselectivities of mutants Y84V, Y84L, Y84I and Y84T are respectively improved to 98.0%, 92.1%, 95.5% and 90.1%.

Therefore, the catalytic performance of the mutant obtained by the invention is greatly improved relative to that of the wild-type olefinic bond reductase OYE2p, wherein the mutant Y84V has the best catalytic activity and the highest stereoselectivity.

Example 4 kinetic parameters of wild type OYE2p and its mutant Y84V

The kinetic parameters of the enzyme ethylenic reductase OYE2p and its mutant forms for citral were determined by fixing the concentration of NADH at 0.2mM under standard conditions and varying the concentration of E-citral or Z-citral between 0.01 and 10 mM. The standard detection method is as follows: the total reaction volume was 200. mu.L, 2-20. mu.g of the pure enzyme obtained in example 2 and 10mM substrate were added, 100mM PBS buffer (pH7.4) was added to make up to 196. mu.L, and after incubating at 30 ℃ for 5min with shaking, 4. mu.L of NADH solution (10mM) was added, and the mixture was immediately placed in a microplate reader to measure the change in absorbance at 340 nm. To ensure the accuracy of the experiment, the assay was repeated three times for each sample.

The results show k for E-citral, wild type and Y84V mutant_cat/K_mThe values are 1.78S respectively^-1/mM^-1And 3.05S^-1/mM^-1. K for Z-citral, wild type and Y84V mutant_cat/K_mThe values are respectively 0.42S^-1/mM^-1And 0.50S^-1/mM^-1。

Catalytic efficiency of mutant Y84V for E-citral and Z-citral (k)_cat/K_mValue) of the amino acid residues ofHas important function.

Example 5 application of wild type OYE2p and its mutant Y84V in catalyzing citral to produce (R) -citronellal

The crude enzyme solution of OYE2p and each mutant expression strain obtained in step 2 of example 2 was freeze-dried to obtain a freeze-dried crude enzyme powder. The reaction system for catalyzing citral by wild type OYE2p and mutant Y84V is as follows: 200mM citral (in DMSO), 0.2mM NAD⁺250mM glucose, 100mM phosphate buffer (pH8.5), 0.15g of crude lyophilized OYE2p or its mutant Y84V enzyme powder, and 0.05g of crude lyophilized Glucose Dehydrogenase (GDH) enzyme powder, in a total volume of 5 mL. The reaction was carried out in a 25mL Erlenmeyer flask under 30 ℃ with shaking at 200 rpm. During the reaction, Na is added₂CO₃The pH value of the solution adjusting system is maintained at 8.5.

As shown in FIG. 2, the wild type OYE2p catalyzed 200mM citral for 10h, the conversion reached 91.6%, but the ee value of the product (R) -citronellal formed was only 87.6%. As shown in FIG. 3, the mutant Y84V obtained by the invention has 100% conversion rate after catalyzing 200mM citral for 10h, and the ee value of the product is more than 95%. Therefore, the catalytic performance of the mutant obtained by the invention is greatly improved compared with that of the wild-type olefinic bond reductase OYE2 p.

The above embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and various changes may be made in the above embodiments of the present invention. All simple and equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the scope of the claims of the present invention. The invention has not been described in detail in order to avoid obscuring the invention.

SEQUENCE LISTING

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His Ser Ile Thr Lys Asp Glu Ile Lys Gln Tyr Val Lys Glu Tyr Val

165 170 175

Gln Ala Ala Lys Asn Ser Ile Ala Ala Gly Ala Asp Gly Val Glu Ile

180 185 190

His Ser Ala Asn Gly Tyr Leu Leu Asn Gln Phe Leu Asp Pro His Ser

195 200 205

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

210 215 220

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

225 230 235 240

Lys Val Gly Leu Arg Leu Ser Pro Tyr Gly Val Phe Asn Ser Met Ser

245 250 255

Gly Gly Ala Glu Thr Gly Ile Val Ala Gln Tyr Ala Tyr Val Leu Gly

260 265 270

Glu Leu Glu Arg Arg Ala Lys Ala Gly Lys Arg Leu Ala Phe Val His

275 280 285

Leu Ile Glu Pro Arg Val Thr Asn Pro Phe Leu Thr Glu Gly Glu Gly

290 295 300

Glu Tyr Asn Gly Gly Ser Asn Glu Phe Ala Tyr Ser Ile Trp Lys Gly

305 310 315 320

Pro Ile Ile Arg Ala Gly Asn Phe Ala Leu His Pro Glu Val Val Arg

325 330 335

Glu Glu Val Lys Asp Pro Arg Thr Leu Ile Gly Tyr Gly Arg Phe Phe

340 345 350

Ile Ser Asn Pro Asp Leu Val Asp Arg Leu Glu Lys Gly Leu Pro Leu

355 360 365

Asn Lys Tyr Asp Arg Asp Thr Phe Tyr Lys Met Ser Ala Glu Gly Tyr

370 375 380

Ile Asp Tyr Pro Thr Tyr Glu Glu Ala Leu Lys Leu Gly Trp Asp Lys

385 390 395 400

Asn

<210> 4

<211> 400

<212> PRT

<213> Artificial sequence

<400> 4

Met Val Pro Phe Val Lys Asp Phe Lys Pro Gln Ala Leu Gly Asp Thr

1 5 10 15

Asn Leu Phe Lys Pro Ile Lys Ile Gly Asn Asn Glu Leu Leu His Arg

20 25 30

Ala Val Ile Pro Pro Leu Thr Arg Met Arg Ala Gln His Pro Gly Asn

35 40 45

Ile Pro Asn Arg Asp Trp Ala Val Glu Tyr Tyr Ala Gln Arg Ala Gln

50 55 60

Arg Pro Gly Thr Leu Ile Ile Thr Glu Gly Thr Phe Pro Ser Pro Gln

65 70 75 80

Ser Gly Gly Val Asn Ala Pro Gly Ile Trp Ser Glu Glu Gln Ile Lys

85 90 95

Glu Trp Thr Lys Ile Phe Lys Ala Ile His Glu Lys Lys Ser Phe Ala

100 105 110

Trp Val Gln Leu Trp Val Leu Gly Trp Ala Ala Phe Pro Asp Thr Leu

115 120 125

Ala Arg Asp Gly Leu Arg Tyr Asp Ser Ala Ser Asp Asn Val Tyr Met

130 135 140

Asn Ala Glu Gln Glu Glu Lys Ala Lys Lys Ala Asn Asn Pro Gln His

145 150 155 160

Ser Ile Thr Lys Asp Glu Ile Lys Gln Tyr Val Lys Glu Tyr Val Gln

165 170 175

Ala Ala Lys Asn Ser Ile Ala Ala Gly Ala Asp Gly Val Glu Ile His

180 185 190

Ser Ala Asn Gly Tyr Leu Leu Asn Gln Phe Leu Asp Pro His Ser Asn

195 200 205

Asn Arg Thr Asp Glu Tyr Gly Gly Ser Ile Glu Asn Arg Ala Arg Phe

210 215 220

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

225 230 235 240

Val Gly Leu Arg Leu Ser Pro Tyr Gly Val Phe Asn Ser Met Ser Gly

245 250 255

Gly Ala Glu Thr Gly Ile Val Ala Gln Tyr Ala Tyr Val Leu Gly Glu

260 265 270

Leu Glu Arg Arg Ala Lys Ala Gly Lys Arg Leu Ala Phe Val His Leu

275 280 285

Ile Glu Pro Arg Val Thr Asn Pro Phe Leu Thr Glu Gly Glu Gly Glu

290 295 300

Tyr Asn Gly Gly Ser Asn Glu Phe Ala Tyr Ser Ile Trp Lys Gly Pro

305 310 315 320

Ile Ile Arg Ala Gly Asn Phe Ala Leu His Pro Glu Val Val Arg Glu

325 330 335

Glu Val Lys Asp Pro Arg Thr Leu Ile Gly Tyr Gly Arg Phe Phe Ile

340 345 350

Ser Asn Pro Asp Leu Val Asp Arg Leu Glu Lys Gly Leu Pro Leu Asn

355 360 365

Lys Tyr Asp Arg Asp Thr Phe Tyr Lys Met Ser Ala Glu Gly Tyr Ile

370 375 380

Asp Tyr Pro Thr Tyr Glu Glu Ala Leu Lys Leu Gly Trp Asp Lys Asn

385 390 395 400

<210> 5

<211> 399

<212> PRT

<213> Artificial sequence

<400> 5

Met Val Pro Phe Val Lys Asp Phe Lys Pro Gln Ala Leu Gly Asp Thr

1 5 10 15

Asn Leu Phe Lys Pro Ile Lys Ile Gly Asn Asn Glu Leu Leu His Arg

20 25 30

Ala Val Ile Pro Pro Leu Thr Arg Met Arg Ala Gln His Pro Gly Asn

35 40 45

Ile Pro Asn Arg Asp Trp Ala Val Glu Tyr Tyr Ala Gln Arg Ala Gln

50 55 60

Arg Pro Gly Thr Leu Ile Ile Thr Glu Gly Thr Phe Pro Ser Pro Gln

65 70 75 80

Ser Gly Gly Leu Ala Pro Gly Ile Trp Ser Glu Glu Gln Ile Lys Glu

85 90 95

Trp Thr Lys Ile Phe Lys Ala Ile His Glu Lys Lys Ser Phe Ala Trp

100 105 110

Val Gln Leu Trp Val Leu Gly Trp Ala Ala Phe Pro Asp Thr Leu Ala

115 120 125

Arg Asp Gly Leu Arg Tyr Asp Ser Ala Ser Asp Asn Val Tyr Met Asn

130 135 140

Ala Glu Gln Glu Glu Lys Ala Lys Lys Ala Asn Asn Pro Gln His Ser

145 150 155 160

Ile Thr Lys Asp Glu Ile Lys Gln Tyr Val Lys Glu Tyr Val Gln Ala

165 170 175

Ala Lys Asn Ser Ile Ala Ala Gly Ala Asp Gly Val Glu Ile His Ser

180 185 190

Ala Asn Gly Tyr Leu Leu Asn Gln Phe Leu Asp Pro His Ser Asn Asn

195 200 205

Arg Thr Asp Glu Tyr Gly Gly Ser Ile Glu Asn Arg Ala Arg Phe Thr

210 215 220

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

225 230 235 240

Gly Leu Arg Leu Ser Pro Tyr Gly Val Phe Asn Ser Met Ser Gly Gly

245 250 255

Ala Glu Thr Gly Ile Val Ala Gln Tyr Ala Tyr Val Leu Gly Glu Leu

260 265 270

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

275 280 285

Glu Pro Arg Val Thr Asn Pro Phe Leu Thr Glu Gly Glu Gly Glu Tyr

290 295 300

Asn Gly Gly Ser Asn Glu Phe Ala Tyr Ser Ile Trp Lys Gly Pro Ile

305 310 315 320

Ile Arg Ala Gly Asn Phe Ala Leu His Pro Glu Val Val Arg Glu Glu

325 330 335

Val Lys Asp Pro Arg Thr Leu Ile Gly Tyr Gly Arg Phe Phe Ile Ser

340 345 350

Asn Pro Asp Leu Val Asp Arg Leu Glu Lys Gly Leu Pro Leu Asn Lys

355 360 365

Tyr Asp Arg Asp Thr Phe Tyr Lys Met Ser Ala Glu Gly Tyr Ile Asp

370 375 380

Tyr Pro Thr Tyr Glu Glu Ala Leu Lys Leu Gly Trp Asp Lys Asn

385 390 395

<210> 6

<211> 398

<212> PRT

<213> Artificial sequence

<400> 6

Met Val Pro Phe Val Lys Asp Phe Lys Pro Gln Ala Leu Gly Asp Thr

1 5 10 15

Asn Leu Phe Lys Pro Ile Lys Ile Gly Asn Asn Glu Leu Leu His Arg

20 25 30

Ala Val Ile Pro Pro Leu Thr Arg Met Arg Ala Gln His Pro Gly Asn

35 40 45

Ile Pro Asn Arg Asp Trp Ala Val Glu Tyr Tyr Ala Gln Arg Ala Gln

50 55 60

Arg Pro Gly Thr Leu Ile Ile Thr Glu Gly Thr Phe Pro Ser Pro Gln

65 70 75 80

Ser Gly Gly Ile Pro Gly Ile Trp Ser Glu Glu Gln Ile Lys Glu Trp

85 90 95

Thr Lys Ile Phe Lys Ala Ile His Glu Lys Lys Ser Phe Ala Trp Val

100 105 110

Gln Leu Trp Val Leu Gly Trp Ala Ala Phe Pro Asp Thr Leu Ala Arg

115 120 125

Asp Gly Leu Arg Tyr Asp Ser Ala Ser Asp Asn Val Tyr Met Asn Ala

130 135 140

Glu Gln Glu Glu Lys Ala Lys Lys Ala Asn Asn Pro Gln His Ser Ile

145 150 155 160

Thr Lys Asp Glu Ile Lys Gln Tyr Val Lys Glu Tyr Val Gln Ala Ala

165 170 175

Lys Asn Ser Ile Ala Ala Gly Ala Asp Gly Val Glu Ile His Ser Ala

180 185 190

Asn Gly Tyr Leu Leu Asn Gln Phe Leu Asp Pro His Ser Asn Asn Arg

195 200 205

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

210 215 220

Glu Val Val Asp Ala Val Val Asp Ala Ile Gly Pro Glu Lys Val Gly

225 230 235 240

Leu Arg Leu Ser Pro Tyr Gly Val Phe Asn Ser Met Ser Gly Gly Ala

245 250 255

Glu Thr Gly Ile Val Ala Gln Tyr Ala Tyr Val Leu Gly Glu Leu Glu

260 265 270

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

275 280 285

Pro Arg Val Thr Asn Pro Phe Leu Thr Glu Gly Glu Gly Glu Tyr Asn

290 295 300

Gly Gly Ser Asn Glu Phe Ala Tyr Ser Ile Trp Lys Gly Pro Ile Ile

305 310 315 320

Arg Ala Gly Asn Phe Ala Leu His Pro Glu Val Val Arg Glu Glu Val

325 330 335

Lys Asp Pro Arg Thr Leu Ile Gly Tyr Gly Arg Phe Phe Ile Ser Asn

340 345 350

Pro Asp Leu Val Asp Arg Leu Glu Lys Gly Leu Pro Leu Asn Lys Tyr

355 360 365

Asp Arg Asp Thr Phe Tyr Lys Met Ser Ala Glu Gly Tyr Ile Asp Tyr

370 375 380

Pro Thr Tyr Glu Glu Ala Leu Lys Leu Gly Trp Asp Lys Asn

385 390 395

<210> 7

<211> 397

<212> PRT

<213> Artificial sequence

<400> 7

Met Val Pro Phe Val Lys Asp Phe Lys Pro Gln Ala Leu Gly Asp Thr

1 5 10 15

Asn Leu Phe Lys Pro Ile Lys Ile Gly Asn Asn Glu Leu Leu His Arg

20 25 30

Ala Val Ile Pro Pro Leu Thr Arg Met Arg Ala Gln His Pro Gly Asn

35 40 45

Ile Pro Asn Arg Asp Trp Ala Val Glu Tyr Tyr Ala Gln Arg Ala Gln

50 55 60

Arg Pro Gly Thr Leu Ile Ile Thr Glu Gly Thr Phe Pro Ser Pro Gln

65 70 75 80

Ser Gly Gly Thr Gly Ile Trp Ser Glu Glu Gln Ile Lys Glu Trp Thr

85 90 95

Lys Ile Phe Lys Ala Ile His Glu Lys Lys Ser Phe Ala Trp Val Gln

100 105 110

Leu Trp Val Leu Gly Trp Ala Ala Phe Pro Asp Thr Leu Ala Arg Asp

115 120 125

Gly Leu Arg Tyr Asp Ser Ala Ser Asp Asn Val Tyr Met Asn Ala Glu

130 135 140

Gln Glu Glu Lys Ala Lys Lys Ala Asn Asn Pro Gln His Ser Ile Thr

145 150 155 160

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

165 170 175

Asn Ser Ile Ala Ala Gly Ala Asp Gly Val Glu Ile His Ser Ala Asn

180 185 190

Gly Tyr Leu Leu Asn Gln Phe Leu Asp Pro His Ser Asn Asn Arg Thr

195 200 205

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

210 215 220

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

225 230 235 240

Arg Leu Ser Pro Tyr Gly Val Phe Asn Ser Met Ser Gly Gly Ala Glu

245 250 255

Thr Gly Ile Val Ala Gln Tyr Ala Tyr Val Leu Gly Glu Leu Glu Arg

260 265 270

Arg Ala Lys Ala Gly Lys Arg Leu Ala Phe Val His Leu Ile Glu Pro

275 280 285

Arg Val Thr Asn Pro Phe Leu Thr Glu Gly Glu Gly Glu Tyr Asn Gly

290 295 300

Gly Ser Asn Glu Phe Ala Tyr Ser Ile Trp Lys Gly Pro Ile Ile Arg

305 310 315 320

Ala Gly Asn Phe Ala Leu His Pro Glu Val Val Arg Glu Glu Val Lys

325 330 335

Asp Pro Arg Thr Leu Ile Gly Tyr Gly Arg Phe Phe Ile Ser Asn Pro

340 345 350

Asp Leu Val Asp Arg Leu Glu Lys Gly Leu Pro Leu Asn Lys Tyr Asp

355 360 365

Arg Asp Thr Phe Tyr Lys Met Ser Ala Glu Gly Tyr Ile Asp Tyr Pro

370 375 380

Thr Tyr Glu Glu Ala Leu Lys Leu Gly Trp Asp Lys Asn

385 390 395

<210> 8

<211> 396

<212> PRT

<213> Artificial sequence

<400> 8

Met Val Pro Phe Val Lys Asp Phe Lys Pro Gln Ala Leu Gly Asp Thr

1 5 10 15

Asn Leu Phe Lys Pro Ile Lys Ile Gly Asn Asn Glu Leu Leu His Arg

20 25 30

Ala Val Ile Pro Pro Leu Thr Arg Met Arg Ala Gln His Pro Gly Asn

35 40 45

Ile Pro Asn Arg Asp Trp Ala Val Glu Tyr Tyr Ala Gln Arg Ala Gln

50 55 60

Arg Pro Gly Thr Leu Ile Ile Thr Glu Gly Thr Phe Pro Ser Pro Gln

65 70 75 80

Ser Gly Gly Cys Ile Trp Ser Glu Glu Gln Ile Lys Glu Trp Thr Lys

85 90 95

Ile Phe Lys Ala Ile His Glu Lys Lys Ser Phe Ala Trp Val Gln Leu

100 105 110

Trp Val Leu Gly Trp Ala Ala Phe Pro Asp Thr Leu Ala Arg Asp Gly

115 120 125

Leu Arg Tyr Asp Ser Ala Ser Asp Asn Val Tyr Met Asn Ala Glu Gln

130 135 140

Glu Glu Lys Ala Lys Lys Ala Asn Asn Pro Gln His Ser Ile Thr Lys

145 150 155 160

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

165 170 175

Ser Ile Ala Ala Gly Ala Asp Gly Val Glu Ile His Ser Ala Asn Gly

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Gly Ile Val Ala Gln Tyr Ala Tyr Val Leu Gly Glu Leu Glu Arg Arg

260 265 270

Ala Lys Ala Gly Lys Arg Leu Ala Phe Val His Leu Ile Glu Pro Arg

275 280 285

Val Thr Asn Pro Phe Leu Thr Glu Gly Glu Gly Glu Tyr Asn Gly Gly

290 295 300

Ser Asn Glu Phe Ala Tyr Ser Ile Trp Lys Gly Pro Ile Ile Arg Ala

305 310 315 320

Gly Asn Phe Ala Leu His Pro Glu Val Val Arg Glu Glu Val Lys Asp

325 330 335

Pro Arg Thr Leu Ile Gly Tyr Gly Arg Phe Phe Ile Ser Asn Pro Asp

340 345 350

Leu Val Asp Arg Leu Glu Lys Gly Leu Pro Leu Asn Lys Tyr Asp Arg

355 360 365

Asp Thr Phe Tyr Lys Met Ser Ala Glu Gly Tyr Ile Asp Tyr Pro Thr

370 375 380

Tyr Glu Glu Ala Leu Lys Leu Gly Trp Asp Lys Asn

385 390 395

<210> 9

<211> 1203

<212> DNA

<213> Artificial sequence

<400> 9

atggttccat ttgttaagga ctttaagcca caagctttgg gtgacaccaa cttattcaaa 60

ccaatcaaaa ttggtaacaa tgaacttcta caccgtgctg tcattcctcc attgactaga 120

atgagagccc aacatccagg taatattcca aacagagact gggccgttga atactacgct 180

caacgtgctc aaagaccagg aaccttgatt atcactgaag gtacctttcc ctctccacaa 240

tctgggggtg ttgacaatgc tccaggtatc tggtccgaag aacaaattaa agaatggacc 300