阅读说明：本技术 一种羧肽酶及其编码基因和应用 (Carboxypeptidase, and coding gene and application thereof ) 是由 叶茂 邓毛程 李静 张远平 尚红岩 于 2021-07-27 设计创作，主要内容包括：本发明公开了一种羧肽酶及其编码基因和应用。该羧肽酶的氨基酸序列如SEQ ID NO.1所示。本发明还提供了编码该羧肽酶的核苷酸序列。该羧肽酶具有能水解几乎所有的氨基酸残基的作用,具有良好的应用潜力,并且对丰富羧肽酶家族成员,开发我国传统调味品环境资源都具有重要意义。因此,可将其用于制备小分子寡肽,可通过如下步骤制备小分子寡肽：将本发明提供的羧肽酶加入到含有蛋白底物的水解反应体系中,在25～35℃条件下进行酶水解反应2～8h,得到相应的小分子寡肽。(The invention discloses a carboxypeptidase, and a coding gene and application thereof. The amino acid sequence of the carboxypeptidase is shown in SEQ ID NO. 1. The invention also provides a nucleotide sequence for coding the carboxypeptidase. The carboxypeptidase has the function of hydrolyzing almost all amino acid residues, has good application potential, and has important significance for enriching members of carboxypeptidase families and developing environmental resources of traditional condiments in China. Thus, it can be used to prepare small molecule oligopeptides, which can be prepared by: the carboxypeptidase provided by the invention is added into a hydrolysis reaction system containing a protein substrate, and the enzyme hydrolysis reaction is carried out for 2-8h at the temperature of 25-35 ℃, so as to obtain the corresponding small molecular oligopeptide.)

1. A carboxypeptidase enzyme, comprising: the amino acid sequence is shown in SEQ ID NO. 1.

2. A nucleotide sequence characterized in that: is a nucleotide sequence encoding the carboxypeptidase of claim 1.

3. The nucleotide sequence of claim 2, characterized in that: the nucleotide sequence is a sequence without introns and with a complete open reading frame of 1365 bp.

4. The nucleotide sequence of claim 3, wherein: the nucleotide sequence is shown as SEQ ID NO. 2.

5. The process for producing the carboxypeptidase of claim 1, comprising the steps of:

(1) cloning the nucleotide sequence of any one of claims 2-4 into an expression vector, and transferring into an expression cell to obtain a cell containing a recombinant vector;

(2) culturing the cell containing the recombinant vector obtained in the step (1), inducing by IPTG, separating and purifying from the culture to obtain the carboxypeptidase.

6. The process for producing a carboxypeptidase of claim 5, wherein:

the expression vector in the step (1) is pET-32a (+);

the expression cell in the step (1) is escherichia coli BL21(DE 3);

the culture medium in the step (2) is LB liquid medium containing 100. mu.g/ml ampicillin.

7. The process for producing a carboxypeptidase of claim 5, wherein:

the specific steps of the induction in the step (2) are as followsThe following: when the cells containing the recombinant vector were grown to OD₆₀₀When the concentration is 0.7-0.9, IPTG is added to the final concentration of 0.5-1.5 mM, and the mixture is cultured for 20-25 h at 20-30 ℃ under the condition of 150-250 r/min.

8. Use of the carboxypeptidase of claim 1 to prepare a small molecule oligopeptide.

9. The use of a carboxypeptidase for preparing a small molecule oligopeptide according to claim 8, comprising the steps of: adding the carboxypeptidase of claim 1 into a hydrolysis reaction system containing a protein substrate, and carrying out an enzymatic hydrolysis reaction for 2-8h at 25-35 ℃ to obtain a corresponding small molecular oligopeptide.

Technical Field

The invention belongs to the field of genetic engineering and enzyme engineering, and particularly relates to carboxypeptidase, and a coding gene and application thereof.

Background

Carboxypeptidases (CPs) refer to a class of exopeptidases that specifically catalyze the hydrolysis of the carboxy-terminal amino acid of polypeptide chains, and are industrial enzyme preparations with significant potential for use. Carboxypeptidases can be classified mainly into serine carboxypeptidases (EC.3.4.16), metallocarboxypeptidases (EC.3.4.17) and cysteine carboxypeptidases (EC.3.4.18), depending on the mechanism of action at the site of the enzyme activity. The application of carboxypeptidase mainly relates to a plurality of disciplines such as biology, chemistry, medicine and the like. For example, in the food and feed industries, the method can be used for preparing high F value oligopeptides, removing ochratoxin, debittering polypeptides, prolonging or specifically modifying bioactive polypeptides; in the field of biology, the method can be used for polypeptide synthesis and polypeptide amino acid sequence determination, and can also be used as a model enzyme to provide help for the research of other enzymes; in medical application, because the carboxypeptidase is widely involved in biochemical reaction of organisms, the purpose of diagnosing and treating diseases can be achieved by detecting the carboxypeptidase in vivo; in addition, the composition can be used for degrading in vivo harmful substances (methotrexate and the like) in medicine. In recent years, new functions and new applications of carboxypeptidase are continuously discovered and developed, so that the carboxypeptidase has higher research value and wider application prospect.

Metagenomics (Metagenomics) is an emerging scientific technology that has emerged in recent years with the rapid development of microbiology and modern life sciences. The basic research idea is to directly extract the genomic DNA of all microorganisms in the environment, clone to a proper vector, construct a metagenomic library, collect the nucleic acid information of all microorganisms in the environment together, and obtain useful enzymes, antibiotics, active substances and the like from the library by using a sequence screening or functional screening mode. Many novel biocatalysts, such as esterases, cellulases, xylanases, beta-glucosidases and the like, have been successfully screened by metagenomic library technology, and many characteristic information about the novel enzymes are obtained on the basis of the biocatalysts.

So far, no relevant report exists at home and abroad in the research of searching a new carboxypeptidase gene from a metagenomic library.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a carboxypeptidase. The enzyme has low specificity to C-terminal amino acid residues, can hydrolyze almost all amino acid residues, and has important significance for enriching carboxypeptidase family members and developing a new way for hydrolyzing protein into flavor peptides.

Another object of the present invention is to provide a gene encoding the carboxypeptidase.

It is a further object of the present invention to provide the use of said carboxypeptidase.

The purpose of the invention is realized by the following technical scheme: a carboxypeptidase enzyme, having the amino acid sequence shown below:

MGSTVTEVKGMFSVRFIAIVTMLTLFLVLDLSAAAEKTGAALDYEANSGKILYQQNADEIIAIASMTKMMSQLEYLVHEAVDKGKIALDQKVKVSEGGYKTSQDIETSNVPENGGRFPNYTVKEYEPMAIFEGNGSATIARLAEGTAGKEVDFVLKMANDHAKEWGGSDICLKKYKFVNATGLTNKDLKGGPEGTTPEKNFEMSALDVAFVFAPQRLNPDGYPVQLDTAKIPGKKEFWRKNPFTSTGGNWMLPGIKQYDGLKWNKTGTSPEAEYKGFTGTVSEPEGETRNISVVIIKTYPSSNTARYYVDTKKHSYLIGGLILNNFEKKMYGTDSSVNFGQETISFDNARDKDVVVQTKQYISLPDQKGSKDVYKKMEFKSKGQEAPIKQGAKLGSMTISKDAEDPGFLSRGKSMKVTTSAIEFANEFTRSMREIGLLFFSGVWNAVDVKNNTVK。

a nucleotide sequence encoding said carboxypeptidase; preferably a nucleotide sequence without introns and with a complete open reading frame of 1365 bp; more preferably a sequence as shown below:

ATGGGTTCTACCGTTACCGAAGTTAAAGGTATGTTCTCTGTTCGTTTCATCGCTATCGTTACCATGCTGACCCTGTTCCTGGTTCTGGACCTGTCTGCTGCTGCTGAAAAAACCGGTGCTGCTCTGGACTACGAAGCTAACTCTGGTAAAATCCTGTACCAGCAGAACGCTGACGAAATCATCGCTATCGCTTCTATGACCAAAATGATGTCTCAGCTGGAATACCTGGTTCACGAAGCTGTTGACAAAGGTAAAATCGCTCTGGACCAGAAAGTTAAAGTTTCTGAAGGTGGTTACAAAACCTCTCAGGACATCGAAACCTCTAACGTTCCGGAAAACGGTGGTCGTTTCCCGAACTACACCGTTAAAGAATACGAACCGATGGCTATCTTCGAAGGTAACGGTTCTGCTACCATCGCTCGTCTGGCTGAAGGTACCGCTGGTAAAGAAGTTGACTTCGTTCTGAAAATGGCTAACGACCACGCTAAAGAATGGGGTGGTTCTGACATCTGCCTGAAAAAATACAAATTCGTTAACGCTACCGGTCTGACCAACAAAGACCTGAAAGGTGGTCCGGAAGGTACCACCCCGGAAAAAAACTTCGAAATGTCTGCTCTGGACGTTGCTTTCGTTTTCGCTCCGCAGCGTCTGAACCCGGACGGTTACCCGGTTCAGCTGGACACCGCTAAAATCCCGGGTAAAAAAGAATTCTGGCGTAAAAACCCGTTCACCTCTACCGGTGGTAACTGGATGCTGCCGGGTATCAAACAGTACGACGGTCTGAAATGGAACAAAACCGGTACCTCTCCGGAAGCTGAATACAAAGGTTTCACCGGTACCGTTTCTGAACCGGAAGGTGAAACCCGTAACATCTCTGTTGTTATCATCAAAACCTACCCGTCTTCTAACACCGCTCGTTACTACGTTGACACCAAAAAACACTCTTACCTGATCGGTGGTCTGATCCTGAACAACTTCGAAAAAAAAATGTACGGTACCGACTCTTCTGTTAACTTCGGTCAGGAAACCATCTCTTTCGACAACGCTCGTGACAAAGACGTTGTTGTTCAGACCAAACAGTACATCTCTCTGCCGGACCAGAAAGGTTCTAAAGACGTTTACAAAAAAATGGAATTCAAATCTAAAGGTCAGGAAGCTCCGATCAAACAGGGTGCTAAACTGGGTTCTATGACCATCTCTAAAGACGCTGAAGACCCGGGTTTCCTGTCTCGTGGTAAATCTATGAAAGTTACCACCTCTGCTATCGAATTCGCTAACGAATTCACCCGTTCTATGCGTGAAATCGGTCTGCTGTTCTTCTCTGGTGTTTGGAACGCTGTTGACGTTAAAAACAACACCGTTAAA。

the nucleotide sequence can be obtained by a chemical synthesis method, and can also be prepared by the following steps:

firstly, extracting an environmental sample of traditional fermented food, extracting a total genome and purifying;

secondly, carrying out enzyme digestion treatment on the purified total genome;

thirdly, connecting the enzyme digestion product to a cloning vector, constructing a metagenome library of the traditional fermented food environment in the escherichia coli engineering bacteria through electric shock transformation, screening carboxypeptidase positive clones from the library by using a SIGEX (substrate-induced gene expression method), and obtaining positive clones through high-throughput screening;

fourthly, designing a primer through sequencing and Blast comparison, and carrying out chain enzyme polymerization reaction by using the plasmid of the positive clone as a template and the designed primer so as to clone and obtain the nucleotide sequence.

The conventional fermented food in the step one comprises fermented bean curd, fermented soya beans, soybean paste, soy sauce and the like.

The enzyme in step two is preferably Sau3 AI.

The cloning vector in the three steps is preferably Puc118 BamHI/BAP.

The Escherichia coli engineering bacteria in the step three are preferably Escherichia coli DH5 alpha.

The SIGEX (substrate-induced gene expression based) approach described in step three was as follows: all the clones were cultured in a solid medium containing an inducer IPTG, expression of carboxypeptidase and fluorescent protein GFP was induced by IPTG, and positive clones expressing GFP with the strongest fluorescence (indirectly indicating the highest expression level of carboxypeptidase) were selected by a flow cytometer (FACS) according to the intensity of GFP fluorescence expression, in which clones expressing GFP must contain the carboxypeptidase operon and the corresponding carboxypeptidase gene.

The designed primers described in step four are as follows:

the upstream primer F1: 5'-CGCGGATCCATGGGTTCTACCGTTAC-3', respectively;

the downstream primer R1: 5'-CGGAAGCTTTTTAACGGTGTTGTT-3' are provided.

In addition, the invention also provides a preparation method of the carboxypeptidase, which can be obtained by a chemical synthesis method or by expression of the nucleotide sequence and comprises the following steps:

(1) cloning the nucleotide sequence into an expression vector, and transferring into an expression cell to obtain a cell containing a recombinant vector;

(2) culturing the cell containing the recombinant vector obtained in the step (1), inducing by IPTG, separating and purifying from the culture to obtain the carboxypeptidase. The carboxypeptidase has the ability to hydrolyze substantially all amino acid residues.

The expression vector in step (1) is preferably pET-32a (+).

The expression cell described in step (1) is preferably E.coli BL21(DE 3).

The medium for the culture described in step (2) is preferably LB liquid medium containing 100. mu.g/ml ampicillin.

The specific steps of the induction in the step (2) are as follows: when the cells containing the recombinant vector were grown to OD₆₀₀When the concentration is 0.7-0.9, adding IPTG to the final concentration of 0.5-1.5 mM, and culturing at 20-30 ℃ for 20-25 h at 150-250 r/min; when the cells containing the recombinant vector were grown to OD₆₀₀When the concentration was 0.8, IPTG was added to a final concentration of 1mM, and the mixture was cultured at 25 ℃ at 200r/min for 22 hours.

The separation in step (2) is preferably performed by centrifugation.

In addition, the invention also provides the application of the carboxypeptidase in preparing small molecular oligopeptides, which preferably comprises the following steps: and adding the carboxypeptidase into a hydrolysis reaction system containing a protein substrate, and carrying out enzymatic hydrolysis reaction for 2-8h at 25-60 ℃ to obtain the corresponding small molecular oligopeptide.

The protein substrate comprises soybean protein, corn protein, wheat protein, yeast protein and the like.

The temperature of the hydrolysis reaction system is preferably 35-55 ℃.

The pH value of the hydrolysis reaction system is 5-9; preferably 6 to 8.

The buffer solution in the hydrolysis reaction system is preferably an acetic acid buffer solution.

The mass concentration of the protein substrate in the hydrolysis reaction system is 1-10%; preferably 2%.

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

(1) compared with the prior art, the carboxypeptidase obtained from the metagenome library of the traditional condiment environment in China by applying the metagenome technology is found to have the function of hydrolyzing almost all amino acid residues, has good application potential, and has important significance for enriching members of carboxypeptidase families and developing the traditional condiment environment resources in China.

(2) According to the invention, through researching the optimal conditions of the enzyme in hydrolyzing protein to generate small molecular oligopeptide, the carboxypeptidase disclosed by the invention is subjected to enzyme catalysis reaction for 2-8h at 25-35 ℃ in the reaction of hydrolyzing protein such as soybean protein, corn protein, wheat protein and yeast protein serving as a substrate raw material to generate small molecular oligopeptide, the mass concentration of the substrate is 1% -10%, the reaction conditions can be adopted to efficiently and quickly hydrolyze to generate small molecular oligopeptide, the molecular weight of the small molecular oligopeptide is less than or equal to 3000Da, and the carboxypeptidase has a good application value.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of carboxypeptidase purification provided by the invention; wherein, the Lane M is a protein Marker; lane 1 is crude recombinant protein enzyme solution (unpurified); lane 2 is the purified protein.

FIG. 2 is a graph showing the results of measurement of the optimum reaction temperature of the purified carboxypeptidase.

FIG. 3 is a graph showing the results of determination of optimum reaction pH of the purified carboxypeptidase.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1 acquisition of carboxypeptidase Gene and validation of proteolytic Generation of Small molecule oligopeptides

1. Obtainment of carboxypeptidase Gene

A fermented food environment metagenome library is constructed from a fermented bean curd fermentation environment sample which is a traditional fermented food in China, and the method comprises the following specific steps: extracting and purifying a total genome; carrying out enzyme digestion treatment on the purified total genome by Sau3 AI; the enzyme-digested product was ligated to pUC118 BamHI/BAP vector, and transformed into E.coli DH5 α by electric shock to construct a conventional fermented food environment metagenomic library.

Culturing all the clones of the obtained genome library in a solid culture medium containing an inducer IPTG, inducing expression of carboxypeptidase and fluorescent protein GFP by using the IPTG, wherein all the clones expressing GFP gene necessarily contain the carboxypeptidase operon and the corresponding carboxypeptidase gene, and selecting a positive clone expressing GFP with strongest fluorescence (indirectly indicating that the expression amount of the carboxypeptidase is highest) by using a flow cytometer (FACS) according to the strength of the fluorescence expression of the GFP.

Extracting the plasmid of the positive clone, sending the plasmid to a sequencing company for sequencing to obtain a nucleic acid sequence of carboxypeptidase, wherein the sequence consists of 1365 basic groups, the nucleic acid sequence is shown as SEQ ID NO.2, and the name is cp _ 215; the polypeptide coded by the nucleic acid contains 455 amino acids, the amino acid sequence of the polypeptide is shown in SEQ ID NO.1, and the polypeptide is named as CP _ 215.

2. Cloning of Gene fragments

Designing an amplification primer according to the sequence of the nucleic acid cp _ 215: the upstream primer F1: 5' -CGCGGATCCATGGGTTCTACCGTTAC-3' (BamHI cleavage site sequence is underlined);the downstream primer R1: 5' -CGGAAGCTTTTTAACGGTGTTGTT-3' (HindIII cleavage site sequence is underlined).

Carrying out PCR amplification by taking plasmids of positive clones as templates and F1 and R1 as primers, wherein the reaction system is as follows: PrimeStar^TM0.2. mu.l of HS DNA Ploymerase (2.5U/. mu.l), 6. mu.l of 5 Xbuffer, 2.4. mu.l of dNTP Mix (2.5mM), 0.5. mu.l of template (100 ng/. mu.l), 0.2. mu.l of each of primers F1 and F2(10mM), and make up to 50. mu.l of ultrapure water. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 2 min; denaturation at 98 deg.C for 10s, annealing at 70 deg.C for 15s, and extension at 72 deg.C for 1.5min for 30 cycles; extension at 72 ℃ for 10min, 4 ℃.

After being purified by a PCR product purification kit, the PCR product is subjected to double digestion for 16h by BamHI and HindIII, is connected with a pET-32a (+) (Invitrogen) expression vector which is treated in the same way, is electrically transformed into an expression host escherichia coli BL21(DE3), positive clones are selected through resistance screening, plasmid DNA is extracted, and sequencing verification is carried out to find that the nucleotide sequence of the plasmid DNA is the same as the sequence in SEQ ID NO.2, so that a target gene fragment can be effectively obtained.

3. Obtaining of recombinant carboxypeptidase CP _215

Inoculating the strain containing the plasmid with the correct sequence verification to LB liquid medium containing 100. mu.g/ml ampicillin, culturing at 30 deg.C and 250r/min for 14h, transferring to 100ml LB liquid medium containing 100. mu.g/ml ampicillin at an inoculum size of 1 (v/v)%, and growing to OD₆₀₀When the concentration is 0.8, adding isopropylthio-beta-D-galactoside to the final concentration of 1.0mM, culturing at 25 ℃ for 22h at 200r/min, centrifuging at 12000r/min for 5min, discarding the supernatant, suspending the thallus in 20ml of 50mM Tri-HCl (pH7.5), crushing the thallus by an ultrasonicator (200w, 5 seconds at intervals, 5 minutes all the way, ice bath), centrifuging at 4 ℃ and 13000r/min for 15min, and collecting the supernatant to obtain the crude enzyme solution of the recombinant carboxypeptidase.

The crude enzyme solution of the recombinant carboxypeptidase is purified into recombinant protein by a His-tag protein Purification kit Proband Purification system of Invitrogen company, and the specific operation steps are carried out according to the product instruction of the company. And (4) quickly freezing the purified recombinant protein by liquid nitrogen, and storing the recombinant protein in an ultralow temperature refrigerator. The concentration of the purified recombinant protein was 180. mu.g/ml.

The results of the measurement of the crude enzyme solution and the purified protein by polyacrylamide gel electrophoresis are shown in FIG. 1. As can be seen, the carboxypeptidase of the invention can be successfully purified from a crude enzyme solution by using a His-tag protein Purification kit Proband Purification system.

4. Verification of function of generating small molecular oligopeptide by hydrolyzing protein

Adding 9ml of pH6.0 acid buffer solution (taking 54.6g of sodium acetate, adding 20ml of 1mol/L acetic acid solution for dissolving, and adding water for diluting to 500 ml) into 1ml or 10 mul of the crude enzyme solution of the recombinant carboxypeptidase to serve as a reaction medium, respectively testing different substrates (such as soybean protein, corn protein, wheat protein, yeast protein and the like) with the concentration of 2 (w/w)%, performing water bath reaction at 30 ℃ for 6h, and hydrolyzing to generate corresponding micromolecule oligopeptides, wherein the molecular weight of the micromolecule oligopeptides is less than or equal to 3000 Da. The product was structurally characterized by GC-MS (gas chromatography-mass spectrometer).

Example 2 determination of optimum reaction temperature of recombinant carboxypeptidase CP _215

Adding 100 μ L of purified enzyme solution into 100mM Tris-HCl buffer (pH7.0) and 1% (w/v) casein as substrate, incubating at 20, 25, 30, 35, 40, 45, 50, 55 and 60 deg.C for 10min, rapidly adding 2ml of 0.4mol/L trichloroacetic acid to terminate the reaction, standing at room temperature for 15min, centrifuging at 1000rpm for 10min, collecting 1ml of supernatant, placing in a test tube, adding 5ml of 0.4mol/L Na₂CO₃Measuring absorbance A of the solution and 1ml of Folin phenol solution_680nm. As shown in FIG. 2, the recombinant carboxypeptidase CP _215 still has high catalytic activity at 35-55 ℃, and the optimal reaction temperature is 40 ℃.

Example 3 determination of optimum reaction pH value of recombinant carboxypeptidase CP _215

Adding 10 mul of purified enzyme solution into 1% (w/v) casein as substrate, respectively keeping the temperature at 40 ℃ for 10min under different pH values of pH 4.0-7.0 (100mM phosphate buffer solution) and pH 7.0-9.0 (Tris-HCl buffer solution), rapidly adding 2ml of trichloroacetic acid with the concentration of 0.4mol/L to terminate the reaction, standing at room temperature for 15min, centrifuging at 1000rpm for 10min, taking 1ml of supernatant, placing the supernatant in a test tube, adding 5ml of 0.4mol/LNa₂CO₃Solutions and1ml of Fulinfen solution, and determination of the light absorption A_680nm. As shown in FIG. 3, the optimum reaction pH of the recombinant carboxypeptidase CP _215 is 7.5, and the enzyme activity is still high at the pH of 5.0-8.5.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> Guangdong institute of light industry and technology

<120> carboxypeptidase, and coding gene and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 455

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> carboxypeptidase

<400> 1

Met Gly Ser Thr Val Thr Glu Val Lys Gly Met Phe Ser Val Arg Phe

1 5 10 15

Ile Ala Ile Val Thr Met Leu Thr Leu Phe Leu Val Leu Asp Leu Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Gly Tyr Lys Thr Ser Gln Asp Ile Glu Thr Ser Asn Val Pro Glu

100 105 110

Asn Gly Gly Arg Phe Pro Asn Tyr Thr Val Lys Glu Tyr Glu Pro Met

115 120 125

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

130 135 140

Gly Thr Ala Gly Lys Glu Val Asp Phe Val Leu Lys Met Ala Asn Asp

145 150 155 160

His Ala Lys Glu Trp Gly Gly Ser Asp Ile Cys Leu Lys Lys Tyr Lys

165 170 175

Phe Val Asn Ala Thr Gly Leu Thr Asn Lys Asp Leu Lys Gly Gly Pro

180 185 190

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

195 200 205

Ala Phe Val Phe Ala Pro Gln Arg Leu Asn Pro Asp Gly Tyr Pro Val

210 215 220

Gln Leu Asp Thr Ala Lys Ile Pro Gly Lys Lys Glu Phe Trp Arg Lys

225 230 235 240

Asn Pro Phe Thr Ser Thr Gly Gly Asn Trp Met Leu Pro Gly Ile Lys

245 250 255

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

260 265 270

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

275 280 285

Arg Asn Ile Ser Val Val Ile Ile Lys Thr Tyr Pro Ser Ser Asn Thr

290 295 300

Ala Arg Tyr Tyr Val Asp Thr Lys Lys His Ser Tyr Leu Ile Gly Gly

305 310 315 320

Leu Ile Leu Asn Asn Phe Glu Lys Lys Met Tyr Gly Thr Asp Ser Ser

325 330 335

Val Asn Phe Gly Gln Glu Thr Ile Ser Phe Asp Asn Ala Arg Asp Lys

340 345 350

Asp Val Val Val Gln Thr Lys Gln Tyr Ile Ser Leu Pro Asp Gln Lys

355 360 365

Gly Ser Lys Asp Val Tyr Lys Lys Met Glu Phe Lys Ser Lys Gly Gln

370 375 380

Glu Ala Pro Ile Lys Gln Gly Ala Lys Leu Gly Ser Met Thr Ile Ser

385 390 395 400

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

405 410 415

Val Thr Thr Ser Ala Ile Glu Phe Ala Asn Glu Phe Thr Arg Ser Met

420 425 430

Arg Glu Ile Gly Leu Leu Phe Phe Ser Gly Val Trp Asn Ala Val Asp

435 440 445

Val Lys Asn Asn Thr Val Lys

450 455

<210> 2

<211> 1365

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence encoding carboxypeptidase

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atgggttcta ccgttaccga agttaaaggt atgttctctg ttcgtttcat cgctatcgtt 60

accatgctga ccctgttcct ggttctggac ctgtctgctg ctgctgaaaa aaccggtgct 120

gctctggact acgaagctaa ctctggtaaa atcctgtacc agcagaacgc tgacgaaatc 180

atcgctatcg cttctatgac caaaatgatg tctcagctgg aatacctggt tcacgaagct 240

gttgacaaag gtaaaatcgc tctggaccag aaagttaaag tttctgaagg tggttacaaa 300

acctctcagg acatcgaaac ctctaacgtt ccggaaaacg gtggtcgttt cccgaactac 360

accgttaaag aatacgaacc gatggctatc ttcgaaggta acggttctgc taccatcgct 420

cgtctggctg aaggtaccgc tggtaaagaa gttgacttcg ttctgaaaat ggctaacgac 480

cacgctaaag aatggggtgg ttctgacatc tgcctgaaaa aatacaaatt cgttaacgct 540

accggtctga ccaacaaaga cctgaaaggt ggtccggaag gtaccacccc ggaaaaaaac 600

ttcgaaatgt ctgctctgga cgttgctttc gttttcgctc cgcagcgtct gaacccggac 660

ggttacccgg ttcagctgga caccgctaaa atcccgggta aaaaagaatt ctggcgtaaa 720

aacccgttca cctctaccgg tggtaactgg atgctgccgg gtatcaaaca gtacgacggt 780

ctgaaatgga acaaaaccgg tacctctccg gaagctgaat acaaaggttt caccggtacc 840

gtttctgaac cggaaggtga aacccgtaac atctctgttg ttatcatcaa aacctacccg 900

tcttctaaca ccgctcgtta ctacgttgac accaaaaaac actcttacct gatcggtggt 960

ctgatcctga acaacttcga aaaaaaaatg tacggtaccg actcttctgt taacttcggt 1020

caggaaacca tctctttcga caacgctcgt gacaaagacg ttgttgttca gaccaaacag 1080

tacatctctc tgccggacca gaaaggttct aaagacgttt acaaaaaaat ggaattcaaa 1140

tctaaaggtc aggaagctcc gatcaaacag ggtgctaaac tgggttctat gaccatctct 1200

aaagacgctg aagacccggg tttcctgtct cgtggtaaat ctatgaaagt taccacctct 1260

gctatcgaat tcgctaacga attcacccgt tctatgcgtg aaatcggtct gctgttcttc 1320

tctggtgttt ggaacgctgt tgacgttaaa aacaacaccg ttaaa 1365

<210> 3

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer F1

<400> 3

cgcggatcca tgggttctac cgttac 26

<210> 4

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer R1

<400> 4

cggaagcttt ttaacggtgt tgtt 24