Protease mutant capable of improving alcohol-soluble protein degradation capacity and coding gene and application thereof

文档序号：824757 发布日期：2021-03-30 浏览：24次中文

阅读说明：本技术 提高降解醇溶蛋白能力的蛋白酶突变体及其编码基因和应用 (Protease mutant capable of improving alcohol-soluble protein degradation capacity and coding gene and application thereof ) 是由肖志壮方安然孙建飞于文君付五兵王金龙于 2021-01-19 设计创作，主要内容包括：本发明提供了提高降解醇溶蛋白能力的蛋白酶突变体及其编码基因和应用。本发明通过使用易错PCR方法对Bacillus licheniformis来源的蛋白酶基因进行改造,再通过筛选得到单点突变的蛋白酶突变体APR01,并在此基础上,利用第二轮易错PCR,筛选获得了两个含有两个点突变的蛋白酶突变体APR02和APR03,以及一个含有三个点突变的蛋白酶突变体APR04。本发明的突变体的降解醇溶蛋白的能力得到明显的提高,能够增加其对醇溶蛋白的降解率,因此具有良好的市场应用前景和工业价值。(The invention provides a protease mutant capable of improving the capability of degrading alcohol soluble protein, and a coding gene and application thereof. The invention uses error-prone PCR method Bacillus licheniformis The source protease gene is transformed, and then the protease mutant APR01 with single point mutation is obtained by screening, and on the basis, two protease mutants APR02 and APR03 with two point mutations and a protease mutant APR04 with three point mutations are obtained by screening by using a second error-prone PCR. The mutant of the invention has obviously improved capability of degrading alcohol soluble protein and can increase the degradation rate of alcohol soluble protein, thus having good market application prospect and industrial value.)

1. A protease mutant APR01 with improved prolamin degradation capability is characterized in that the amino acid sequence of the protease mutant APR01 is shown in SEQ ID NO: 5 is shown in the specification; the protease mutant APR01 is prepared from amino acid sequence SEQ ID NO: 1 from serine at position 234 of the protease to tryptophan.

2. The coding gene of the protease mutant APR01 of claim 1, wherein the nucleotide sequence of the coding gene of the protease mutant APR01 is shown as SEQ ID NO: and 6.

3. A protease mutant APR02 with improved prolamin degradation capability is characterized in that the amino acid sequence of the protease mutant APR02 is shown in SEQ ID NO: 7 is shown in the specification; the protease mutant APR02 is prepared from amino acid sequence SEQ ID NO: 1 from the protease at position 234 from serine to tryptophan and at position 263 from serine to isoleucine.

4. The coding gene of the protease mutant APR02, which is characterized in that the nucleotide sequence of the coding gene of the protease mutant APR02 is shown as SEQ ID NO: shown in fig. 8.

5. A protease mutant APR03 with improved prolamin degradation capability is characterized in that the amino acid sequence of the protease mutant APR03 is shown in SEQ ID NO: 9 is shown in the figure; the protease mutant APR03 is prepared from amino acid sequence SEQ ID NO: 1 from serine at position 234 to tryptophan and glycine at position 264 to tryptophan.

6. The coding gene of the protease mutant APR03 of claim 5, wherein the nucleotide sequence of the coding gene of the protease mutant APR03 is shown as SEQ ID NO: shown at 10.

7. A protease mutant APR04 with improved prolamin degradation capability is characterized in that the amino acid sequence of the protease mutant APR04 is shown in SEQ ID NO: 11 is shown in the figure; the protease mutant APR04 is formed by amino acid sequence SEQ ID NO: 1 from valine to alanine at position 53, tyrosine to histidine at position 161 and serine to tryptophan at position 234.

8. The coding gene of the protease mutant APR04, which is characterized in that the nucleotide sequence of the coding gene of the protease mutant APR04 is shown as SEQ ID NO: shown at 12.

9. A recombinant strain comprising the gene encoding the protease mutant APR01 of claim 2, the gene encoding the protease mutant APR02 of claim 4, the gene encoding the protease mutant APR03 of claim 6, or the gene encoding the protease mutant APR04 of claim 8.

10. Use of the protease mutant APR01 of claim 1, or the protease mutant APR02 of claim 3, or the protease mutant APR03 of claim 5, or the protease mutant APR04 of claim 7 for the preparation of a prolamin-degrading biological agent.

Technical Field

The invention belongs to the field of gene engineering and enzyme engineering, and particularly relates to a protease mutant capable of improving the alcohol soluble protein degradation capacity, and a coding gene and application thereof.

Background

Proteases are enzymes which catalyze the hydrolysis of peptide bonds in proteins, are widely present in animals, plants and microorganisms, have many different physiological functions, and are the first and most mature ones for the development of enzymology. The application of protease in various fields, such as food industry, brewing, detergent industry, feed industry, tanning industry, silk industry, pharmaceutical industry and the like, is more closely related to our life, and the environment puts higher demands on the yield and the characteristic improvement of protease.

The prolamin is one of the components of plant seed storage protein, is insoluble in water, and can be dissolved in 60-95% ethanol solution. The alcohol soluble protein is protein with high content in cereal protein powder, and raw materials such as corn, wheat and sorghum are used in a large amount in the feed industry, wherein the alcohol soluble protein in the corn accounts for about 65% of the total amount of the protein, the alcohol soluble protein in the wheat accounts for about 40% of the total amount of the protein, and the alcohol soluble protein in the sorghum accounts for about 77-82% of the total amount of the protein. The alcohol soluble protein can not be digested by endogenous enzyme due to the unique property, so that the utilization rate of the feed protein is greatly reduced, the economic benefit of cultivation is influenced, and meanwhile, the undigested protein can cause environmental nitrogen pollution when being discharged into the environment through livestock and poultry manure.

The error-prone PCR technology is that when DNA polymerase is used for PCR reaction amplification of a target fragment, mutation frequency in the amplification process is increased by adjusting reaction conditions, so that mutation is randomly introduced into a target gene at a certain frequency, a mutant library is constructed, and a required forward mutant is screened. The error-prone PCR technique can be well applied to molecular modification of proteins.

Disclosure of Invention

The invention provides a protease mutant capable of improving the capability of degrading alcohol soluble protein, and a coding gene and application thereof. The invention relates to a method for constructing a mutant library and performing directional screeningBacillus licheniformis The WX-02 source protease gene is improved, and the mutant with the alcohol soluble protein degradation rate improved is obtained by screening, so that the utilization rate of feed protein is improved, the economic benefit is improved, and the environmental pollution is reduced.

In order to achieve the purpose of the invention, the invention is realized by adopting the following technical scheme:

the invention provides a protease mutant APR01 capable of improving the capability of degrading alcohol soluble protein, wherein the amino acid sequence of the protease mutant APR01 is shown as SEQ ID NO: 5 is shown in the specification; the protease mutant APR01 is prepared from amino acid sequence SEQ ID NO: 1 from serine at position 234 of the protease to tryptophan.

The invention also provides the coding gene of the protease mutant APR01, and the nucleotide sequence of the coding gene of the protease mutant APR01 is shown as SEQ ID NO: and 6.

The invention also provides a protease mutant APR02 capable of improving the capability of degrading alcohol soluble protein, wherein the amino acid sequence of the protease mutant APR02 is shown as SEQ ID NO: 7 is shown in the specification; the protease mutant APR02 is prepared from amino acid sequence SEQ ID NO: 1 from the protease at position 234 to tryptophan and at position 263 to isoleucine.

The invention also provides the coding gene of the protease mutant APR02, and the nucleotide sequence of the coding gene of the protease mutant APR02 is shown as SEQ ID NO: shown in fig. 8.

The invention also provides a protease mutant APR03 capable of improving the capability of degrading alcohol soluble protein, wherein the amino acid sequence of the protease mutant APR03 is shown as SEQ ID NO: 9 is shown in the figure; the protease mutant APR03 is prepared from amino acid sequence SEQ ID NO: 1 from serine at position 234 to tryptophan and glycine at position 264 to tryptophan.

The invention also provides the coding gene of the protease mutant APR03, and the nucleotide sequence of the coding gene of the protease mutant APR03 is shown as SEQ ID NO: shown at 10.

The invention also provides a protease mutant APR04 capable of improving the capability of degrading alcohol soluble protein, wherein the amino acid sequence of the protease mutant APR04 is shown as SEQ ID NO: 11 is shown in the figure; the protease mutant APR04 is formed by amino acid sequence SEQ ID NO: 1 from valine to alanine at position 53, tyrosine to histidine at position 161 and serine to tryptophan at position 234.

The invention also provides the coding gene of the protease mutant APR04, and the nucleotide sequence of the coding gene of the protease mutant APR04 is shown as SEQ ID NO: shown at 12.

The invention also provides a recombinant strain containing the coding gene of the protease mutant APR01, the coding gene of the protease mutant APR02, the coding gene of the protease mutant APR03 or the coding gene of the protease mutant APR 04.

The invention also provides application of the protease mutant APR01, the protease mutant APR02, the protease mutant APR03 or the protease mutant APR04 in preparation of a biological preparation for degrading prolamin.

Compared with the prior art, the invention has the advantages and the technical effects that:

the invention is provided byBacillus licheniformis The WX-02 source protease gene is used as a basis, an error-prone PCR method is used for modifying the gene, then a prolamin substrate plate is used for screening to obtain a single-site mutant APR01 containing S234W, and on the basis of a protease mutant APR01, a second error-prone PCR is used for obtaining double-site mutants APR02 and APR03 containing S234W/S263I and S234W/G264W and a triple-site mutant APR04 containing V53A/Y161H/S234W. The modified mutants of APR01, APR02, APR03 and APR04 have prolamin degradation rates respectively increased by 15.5%, 20.4%, 35.6% and 32.0% compared with that of the original protease, so that the prolamin degradation rates are remarkably increased, and the prolamin degradation capability of the mutants is remarkably improved. Therefore, the prolamin degradation rate of the protease mutant obtained by the technical scheme of the invention is greatly improved compared with that of the wild type, so that the prolamin degradation rate of the protease mutant in feed is improvedAnd the method has good application potential in the fields of food or washing and the like, and has good market application prospect.

Drawings

FIG. 1 is a flat panel diagram of screening for protease mutants of the present invention.

FIG. 2 shows the effect of the protease mutants of the present invention on the dry matter disappearance of zein after enzymatic hydrolysis.

FIG. 3 shows the fermentation data of the protease mutants of the present invention in a 30L fermenter.

Detailed Description

In order to facilitate understanding of the invention, the invention will be described in more detail below with reference to the accompanying drawings and examples, but the scope of the invention is not limited to the following specific examples. The molecular biological experimental methods not specifically described in the following examples can be carried out by referring to specific methods listed in molecular cloning experimental Manual (third edition) written by J. Sambruka (Sambrook) et al, or according to kits and product instructions; reagents and biomaterials used in specific examples are commercially available without specific recitation.

1. Strains and vectors

Bacillus subtilis WB600, plasmid pUB110, Escherichia coli BL21-DE3, plasmid pET-21a (+) were purchased from Invitrogen.

2. Reagents and culture media

Plasmid extraction kit, fragment purification recovery kit, restriction enzyme and the like are purchased from precious bioengineering (Dalian) Co., Ltd; the GeneMorph II random mutation PCR kit was purchased from Stratagene; ampicillin, IPTG, etc. were purchased from Biotechnology engineering (Shanghai) Co., Ltd; protein Marker: blue Plus II Protein Marker (14-120 kDa) was purchased from Beijing Quanjin Biotechnology, Inc.

LB culture medium: 1% tryptone, 0.5% yeast extract, 1% NaCl.

Example 1: error-prone PCR construction of protease mutant library

Reference toBacillus licheniformis Amino acid sequence (SEQ ID NO: 1) of WX-02-derived protease and DNA sequence (SEQ ID NO: 1) thereofID NO: 2) primers were designed, Xba I restriction sites were designed at the 5 'end, and BamH I restriction sites were designed at the 3' end.

The PCR was performed using GeneMorph II random mutation PCR kit with SEQ ID NO: 2 as template, and carrying out random mutation by using the following primer sequences:

BLAPR-F：GCTCTAGAATGATGAGGAAGAAATC（SEQ ID NO：3）；

BLAPR-R：CGCGGATCCTTACTGTGCAGCTGCTTCGA（SEQ ID NO：4）；

the EcoR I and Not I cleavage sites are underlined, respectively.

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 60s and extension at 72 ℃ for 5min for 30 cycles.

And carrying out double enzyme digestion on the amplified multiple random mutation PCR products by using Xba I and BamH I, purifying and recycling the products, connecting the products to a pET-21a (+) vector, transforming escherichia coli BL21-DE3, coating the escherichia coli BL21-DE3 on an ampicillin resistance LB plate, screening positive clones to obtain multiple recombinant vectors pET-APRx and recombinant strains BL21-APRx, and respectively numbering the vectors.

The synthesized nucleotide sequence shown as SEQ ID NO: 2 is connected to pET-21a (+) vector and transformed into Escherichia coli BL21-DE3 to obtain recombinant vector pET-APR0 and recombinant strain BL21-APR 0.

The single colony of the positive recombinant strain BL21-APRx obtained by screening is inoculated to a 96-hole deep-well plate, and the single colony of 2 recombinant strains BL21-APR0 (expressing APR 0) is simultaneously inoculated to each plate to serve as a control. Loading 300 mul LB liquid culture medium (containing 100 mul g/mL ampicillin) into each hole, shaking and culturing at 37 ℃ and 200rpm for 4 hours, transferring 50 mul bacterial liquid to a new 96-hole plate for seed preservation, adding 200 mul LB-Amp culture medium containing IPTG into the residual bacterial liquid of the plate to ensure that the final concentration of IPTG is 1mM and the final concentration of ampicillin is 100 mul g/mL, and shaking and culturing at 37 ℃ and 200rpm for 10 hours to induce and express protease; and (3) repeatedly freezing and thawing the induced bacterial liquid for crushing, centrifuging the crushed cell liquid, and taking supernatant, namely the crude enzyme liquid for later use.

Screening of protease mutants using prolamin substrate plates:

(1) weighing 1g of zein powder, and dissolving in 5mL of 60% ethanol solution;

(2) adding the solution obtained in the step (1) into melted 100mL LB solid medium, quickly mixing uniformly, and pouring into a flat plate;

(3) after the flat plate is solidified, punching holes in the flat plate by using a puncher;

(4) 50-70. mu.L of the crude enzyme solution obtained in example 1 may be added to each well of the plate;

(5) putting the prolamin substrate plate into an incubator at 37 ℃ for overnight culture;

(6) the size of the hydrolysis ring diameter (cm) is observed, and the alcohol soluble protein enzymolysis effect is preliminarily evaluated.

As shown in FIG. 1, the hydrolysis cycle of the crude enzyme solution of No. 6 was found to be significantly larger than that of APR0, indicating that the enzymolysis effect of the hydrolysis cycle is better than that of the control APR0, thereby screening a protease mutant with improved degradation effect on prolamin. And (3) carrying out gene sequencing on the mutant to obtain a mutant which takes APR0 as a starting template and contains S234W single mutation, wherein the mutant is named as APR01, and the amino acid sequence of the mutant is shown as SEQ ID NO: 5, the nucleotide sequence of the coding gene is shown as SEQ ID NO: and 6. The protease mutant APR01 has improved prolamin degradation effect, and the mutation is proved to improve the prolamin degradation capability of the protease.

The mutation pattern of APR01 was S234W (serine S at position 234 to tryptophan W).

Example 2: second round error-prone PCR construction of a mutant library of protease APR01

The coding gene of the protease mutant APR01 screened in the example 1 is used as a template, and the second round of random mutation, the construction process of a mutation library, the use of material reagents, the operation conditions and the like are the same as the example 1; during mutant culture and screening, APR01 is used as a control, and after the test and detection of the degradation rate of the prolamin, the gene sequencing is carried out on the protease mutant with obviously improved degradation rate of the prolamin. Finally, 3 mutants with improved prolamin degradation rate are screened in the round, and are respectively named as APR02, APR03 and APR 04:

the APR02 mutation mode is S234W/S263I (serine S at position 234 is changed into tryptophan W and serine S at position 263 is changed into isoleucine I), and the amino acid sequence is shown in SEQ ID NO: 7, the nucleotide sequence of the coding gene is shown as SEQ ID NO: 8 is shown in the specification;

the mutation mode of the APR03 is S234W/G264W (serine S at position 234 is changed into tryptophan W and glycine G at position 264 is changed into tryptophan W), and the amino acid sequence of the mutation mode is shown as SEQ ID NO: 9, the nucleotide sequence of the coding gene is shown as SEQ ID NO: 10 is shown in the figure;

the mutation mode of the APR04 is V53A/Y161H/S234W (valine V at position 53 is mutated into alanine A, tyrosine Y at position 161 is mutated into histidine H, and serine S at position 234 is mutated into tryptophan W), and the amino acid sequence of the mutation mode is shown as SEQ ID NO: 11, and the nucleotide sequence of the coding gene is shown as SEQ ID NO: shown at 12.

Example 3: expression of protease mutant in bacillus subtilis and zein enzymolysis experiment

The protease mutants APR01, APR02, APR03 and APR04 are cloned into Xba I and BamH I sites of plasmid pUB110 respectively, and the constructed recombinant plasmid is transformed into Bacillus subtilis WB600 by the classical Spizzen method to obtain the recombinant strain of the mutant. And (3) transferring the transformants with the 4 genes into a fermentation medium (50-80 g/L of soybean meal, 60-100g/L of corn starch, 2-4g/L of disodium hydrogen phosphate, 1-2g/L of sodium carbonate and natural pH) to ferment for 78h in a shake flask, centrifuging a culture solution to obtain a supernatant, measuring the average enzyme activity of the fermentation supernatant of each mutant, then obtaining the fermentation supernatant of the transformant with the highest enzyme activity in each mutant, and detecting the degradation effect of the transformant on prolamin.

Accurately weighing 1.00 g of zein, putting the zein into a 100mL triangular flask, adding 25mL of phosphate buffer solution with pH of 8.0, respectively adding 4 protease mutants with the same enzyme activity (1000U is added to 1g of zein) into a test group, and adding inactivated alkaline protease into a control group (boiling water bath for 5 min); adjusting the pH value of the solution to 7.0, then placing the solution into a constant-temperature water bath kettle at 40 ℃, starting a water bath kettle oscillator to continuously oscillate at 120 rpm/min, timing after 5min, and digesting for 4 h. After digestion, well digested zein in the triangular flask is pumped and filtered by a suction pump, and a suction filtered sample is dried to constant weight at 105 ℃. From the dry matter weight G1, the dry matter disappearance was calculated as (G0-G1)/G0X 100% (G0 is the original weight in G).

The enzymolysis results are shown in fig. 2, and the degradation rates of the modified protease mutants of APR01, APR02, APR03 and APR04 to the prolamin are respectively improved by 15.5%, 20.4%, 35.6% and 32.0% compared with the degradation rates of the original protease.

The results show that mutation of Ser at position 234 of APR0 into Trp can improve the degradation effect on prolamin while keeping the original enzyme activity; on the basis, the 263 th site Ser is mutated into Ile to obtain a mutant, or the 264 th site Gly is mutated into Trp to obtain a mutant; or mutation of Val at the 53 th position of the prolamine to Ala and mutation of Tyr at the 161 th position of the prolamine to His to obtain the mutant, and the prolamine degradation effect is further improved.

Example 4: protease mutants were fermented and prepared in a 30L fermentor

Recombinant strains expressing protease mutants APR0, APR01, APR02, APR03 and APR04 were streaked on LB plates containing kanamycin resistance (20. mu.g/mL final concentration), cultured at 37 ℃ until single colonies grew out, single colonies with good growth were selected and streaked on LB plates containing kanamycin resistance (20. mu.g/mL final concentration), the colonies of the recombinant Bacillus subtilis thus activated for three generations were inoculated in 50mL of LB medium containing kanamycin (20. mu.g/mL final concentration), cultured at 37 ℃ and 200rpm for 24 hours, and then inoculated in 1L of LB medium containing kanamycin (20. mu.g/mL final concentration) at an inoculum size of 2%, cultured at 37 ℃ and 200rpm until OD is reached₆₀₀About 5, and is used as a seed liquid inoculation fermentation tank.

The fermentation production process comprises the following steps: 5-10% of soybean meal, 1-5% of corn flour, 1-1.0% of PPG-200000.1, 0.1-1.0% of protease, 0.1-1.0% of amylase, 0.2-0.5% of disodium hydrogen phosphate (12 water), natural pH, 37 ℃, stirring speed of 600rpm, ventilation volume of 1.5 (v/v), and dissolved oxygen controlled at more than 20%. The pH value is natural in the fermentation process, the enzyme activity is measured after fermentation is carried out for 24 hours, and after the fermentation is finished (generally 48 hours), the fermentation liquor is processed by a plate-and-frame filter to obtain crude enzyme liquid, and the crude enzyme liquid is sprayed and dried by a spray tower to form a powder preparation for application test.

The results are shown in fig. 3, which shows that the protease mutants of APR01, APR02, APR03 and APR04 maintain the enzyme activity of the original protease, and the mutants also have a significant improvement on the prolamin degradation rate, so the protease mutants of APR01, APR02, APR03 and APR04 have good application potential.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Sequence listing

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