CN115161307B

CN115161307B - Specific carboxypeptidase for preparing high F value oligopeptide from aspergillus oryzae

Info

Publication number: CN115161307B
Application number: CN202210526163.8A
Authority: CN
Inventors: 熊科; 刘俊兰; 甄红敏; 郑玲燕; 李梦梦; 胡雨萌; 靳雯
Original assignee: Beijing Technology and Business University
Current assignee: Beijing Technology and Business University
Priority date: 2022-05-16
Filing date: 2022-05-16
Publication date: 2023-04-21
Anticipated expiration: 2042-05-16
Also published as: CN115161307A

Abstract

The invention belongs to the technical field of genetic engineering, and provides a specific carboxypeptidase for preparing high-F-value oligopeptide from aspergillus oryzae. The amino acid sequence of the carboxypeptidase is shown as SEQ ID NO.1, and the carboxypeptidase is derived from Aspergillus oryzaeAspergillus oryzae) M30011 with preservation number of CGMCC No.12371. The invention also comprises mutants of the carboxypeptidase, and the amino acid sequence of the mutants is shown in any one of SEQ ID NO. 2-4. The carboxypeptidase can be used to prepare high F oligopeptides. The carboxypeptidase and engineering bacteria for producing the carboxypeptidase are provided by the invention. By modifying special sites, the efficiency of cutting off aromatic amino acids in polypeptide substrates by carboxypeptidase is improved, the F value of the product can be improved, the production cost of an enzyme method is reduced, the energy consumption in the later purification process is reduced, the environmental pollution is reduced, and the industrial production application of efficiently preparing the high F value oligopeptide is realized.

Description

Specific carboxypeptidase for preparing high F value oligopeptide from aspergillus oryzae

Technical Field

The invention belongs to the field of genetic engineering, and in particular relates to carboxypeptidase derived from aspergillus oryzae for preparing high-F-value oligopeptide and engineering bacteria thereof.

Background

Aspergillus oryzaeAspergillus oryzae) Is a safe filamentous fungus, and the aspergillus oryzae is different from the aspergillus flavus although the aspergillus oryzae and the aspergillus flavus belong to the aspergillus flavus genus. It does not produce aflatoxin, and its safety is proved by long-term use in the food industry. Aspergillus oryzaeAspergillus oryzae) The distribution is very wide, and the strain is mainly used in grains, fermented foods, putrefying organic matters, soil and the like, and is a production strain for traditional brewed food sauce and soy sauce in China. It has a long history of use in fermented soya beans, soy sauce, traditional fermented soya bean products and the brewing industry, and is one of 40 safe brewing strains published by the U.S. Food and Drug Administration (FDA) in 1989. Serine carboxypeptidase in Saccharomyces cerevisiae is currently the most widely studied. The sequence was found to be derived from Aspergillus oryzae RIB40 serine carboxypeptidase by NCBI homology alignment.

Modern nutrition research finds that after the human body intakes protein and is acted by digestive enzyme, the protein is absorbed not only in the form of amino acid, but also in the form of small molecular peptide in most cases, and the oligopeptide is absorbed more quickly than free amino acid, which indicates that the biological value and the nutritional utility of the oligopeptide are higher. The high F oligopeptide is a short peptide consisting of 2-10 amino acids, the molar ratio of Branched Chain Amino Acids (BCAA) to Aromatic Amino Acids (AAA) being greater than 20. Meanwhile, the high-F-value oligopeptide is a functional oligopeptide, for example, the high-F-value oligopeptide has the effects of resisting fatigue, resisting oxidization and resisting aging and has the effects of preventing hypertension and liver repair. Compared with acid or alkaline hydrolysis, the enzymatic hydrolysis method has the advantages of mild process conditions, little or no adverse side reaction or product, less salt content of the neutralized final product, capability of controlling the functionality of the final product by selecting specific enzymes and reaction factors, and the like, so that the bioactive peptide is prepared by adopting the enzymatic method. The process of obtaining bioactive peptide through separating and purifying protein from animal and plant is usually used as substrate and is hydrolyzed with proteinase under proper condition. In the process of preparing the high F value oligopeptide by using the enzyme method, the problems of poor enzyme digestion specificity, more enzyme combinations and the like exist. Therefore, there is an urgent need to use a peptidase having a certain specificity for aromatic amino acids to efficiently cleave aromatic amino acids and increase the F value of the product. The cost for preparing the oligopeptide by adopting protease hydrolysis is low, and the value of the product can be effectively improved particularly when a few grain and oil processing byproducts are subjected to enzymolysis, so that the method has great development prospect.

Disclosure of Invention

Aiming at the problems existing in the enzymatic preparation of high-F-value oligopeptide, the invention provides serine carboxypeptidase and engineering bacteria which are derived from Aspergillus oryzae and have specificity to aromatic amino acid, and the high-F-value oligopeptide can be efficiently obtained.

In order to achieve the above purpose, the present invention adopts the following technical scheme.

A carboxypeptidase whose amino acid sequence is shown in SEQ ID NO.1 is derived from Aspergillus oryzaeAspergillus oryzae) M30011 with preservation number of CGMCC No.12371.

The amino acid sequence of the mutant of the carboxypeptidase is shown in any one of SEQ ID NO. 2-4;

wherein,,

SEQ ID NO. 2 is that tyrosine at 271 of the sequence shown in SEQ ID NO.1 is mutated into arginine;

the SEQ ID NO. 3 is that the isoleucine at 464 th site of the sequence shown in SEQ ID NO.1 is mutated into glycine;

SEQ ID NO. 4 is the mutation of methionine at position 517 of the sequence shown in SEQ ID NO.1 to arginine.

A nucleotide encoding the above carboxypeptidase.

A plasmid or engineering bacterium comprising the above-described coding nucleotide; preferably, the engineering bacteria are selected from escherichia coli.

An application of the engineering bacteria in preparing carboxypeptidase.

The application of the carboxypeptidase in preparing the oligopeptide with high F value.

The invention has the following advantages:

the invention provides a genetically modified carboxypeptidase and engineering bacteria for producing the same. By modifying special sites, the efficiency of cutting off aromatic amino acids in polypeptide substrates by carboxypeptidase is improved, the F value of the product can be improved, the production cost of an enzyme method is reduced, the energy consumption in the later purification process is reduced, the environmental pollution is reduced, and the industrial production application of efficiently preparing the high F value oligopeptide is realized.

Biological preservation information

Aspergillus oryzaeAspergillusoryzae) M30011 is preserved in China general microbiological culture Collection center (CGMCC) for 15 days of 2016 at 04 th, and has a preservation address of Beijing, north Chen Silu No.1, national academy of sciences of China, and a preservation number of CGMCC No.12371.

Drawings

FIG. 1 is a gel electrophoresis picture of a CPY gene PCR product;

FIG. 2 is a photograph of a linearized gel electrophoresis of a double-digested pCold TF vector;

FIG. 3 is a photograph of gel electrophoresis of DH 5. Alpha. Competent cells transformed with recombinant plasmid;

FIG. 4 is a map of pCold TF-CPY recombinant plasmid;

FIG. 5 is a gel electrophoresis picture of competent cells of recombinant plasmid transformed BL21 (DE 3);

FIG. 6 is an SDS-PAGE electrophoresis of a crude enzyme solution of CPY enzyme;

FIG. 7 is an agarose electrophoresis diagram during construction of mutants;

FIG. 8 shows SDS-PAGE of crude enzyme solution of the proenzyme and the mutant enzyme (1. Lane is the proenzyme, 2 and 3 lanes are Y271R,4 and 5 lanes are I464G,6 and 7 lanes are M517R);

FIG. 9 shows SDS-PAGE of purified primordial enzyme (crude enzyme solution in

lanes

1, 2, 50mM imidazole in

lanes

3, 4, 100mM imidazole in

lanes

5, 6, 7, 200mM imidazole in

lanes

8, 9, 10, 11);

FIG. 10 is a SDS-PAGE electrophoresis of the purified mutant enzyme;

FIG. 11 shows the F values of oligopeptides obtained by treatment with different enzyme combinations.

Detailed Description

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

EXAMPLE 1M 30011 Strain CPY Gene cloning and expression

Strain M30011 was inoculated into liquid fermentation medium according to the method in CN106190857a, cultured at 30 ℃ for 48h at 200r/min, and after bacterial cells were collected with sterilized filter paper, the residual medium was minimized, and ground with liquid nitrogen pre-cooling. The kit extracts total RNA (A260/A280 ratio 1.95) and synthesizes cDNA.

The primers shown in Table 1 were designed and synthesized, PCR amplification was performed, and a reaction system was established: 10 XTaq Buffer 10. Mu.L, dNTP (10 mM) 2.5. Mu.L, primer CPY-EcoRI-F2. Mu.L, primer CPY-XbaI-R2. Mu.L, taq (5U/. Mu.L) 1. Mu.L, target gene 2. Mu.L, double distilled water to 50. Mu.L. The PCR amplification conditions were: pre-denaturation at 94℃for 3min, then denaturation at 94℃for 30s, annealing at 62.5℃for 30s, extension at 72℃for 1min,34 cycles, extension at 72℃for 5min.

TABLE 1 cloning primer sequence of CPY Gene with cleavage site

The product was subjected to 1% agarose gel electrophoresis and the results are shown in FIG. 1: the size of the product is about 1600bp, the expected target band is recovered by using a gel recovery kit and sequenced, and the obtained target fragment is proved to be the CPY gene with the enzyme cutting site. The CPY gene is obtained by double enzyme digestion and purification of EcoRI and XbaI, and the nucleotide sequence is shown as SEQ ID NO. 7.

Plasmids of pCold TF vector were extracted, digested with EcoRI and XbaI, subjected to 1% agarose gel electrophoresis (FIG. 2), and then the linear plasmids were recovered. Then, the CPY gene and the linear plasmid were ligated and reacted at 37℃for 30 minutes in the following reaction system, and cooled down to 4℃or immediately placed on ice. The reaction system is as follows: 5 XCEIIBuffer 4. Mu.L, exnaseII 2. Mu.L, pCold TF vector 3.3. Mu.L (35 ng/. Mu.L), CPY gene 1.3. Mu.L (48.1 ng/. Mu.L), double distilled water was added to 20. Mu.L.

The plasmids obtained in the above steps are respectively heat-shock-introduced at 42 ℃ for 60 secondsE.coliDH5 alpha competent cells were plated on LB plates containing ampicillin, incubated at 37℃for 12-16h, and after single colony growth was picked up, colony P was performed using CPY gene-specific primers as shown in Table 2CR amplification, screening positive clones.

TABLE 2 PCR amplification of CPY Gene-specific primer sequences

The PCR amplification conditions were: pre-denaturing at 94℃for 5min, then denaturing at 94℃for 30s, annealing at 61.4℃for 30s, extending at 72℃for 1min,34 cycles, and extending at 72℃for 5min to give a fragment of about 1600bp, which was detected by agarose electrophoresis as shown in FIG. 3. The products were sequenced and aligned on NCBI website and the correct recombinant plasmid was named pCold TF-CPY and the pCold TF-CPY profile is shown in FIG. 4.

Plasmid is extracted from positive clones, BL21 (DE 3) competent cells are introduced by heat shock at 42 ℃ for 60 seconds, PCR verification is carried out on the selected bacteria by the primers in the table 2 under the same conditions after the bacteria are cultured for 12-16 hours at 37 ℃ on a coated plate, a fragment with the size of 1600bp is obtained by agarose electrophoresis detection of the product as shown in figure 5, and the sequencing is correct, which indicates that positive transformants are obtained.

Picking up the activated bacterial liquid of the successfully verified transformant in 50mL of ampicillin-resistant liquid LB culture medium with 1% of inoculum size, achieving the purpose of expanding culture, and carrying out OD (optical density) on the transformant at 37 ℃ and 200r/min ₆₀₀ Culturing to 0.6-0.8. And adding IPTG to the fermentation broth to obtain a final concentration of 1.0mM, and continuously carrying out shaking culture at 20 ℃ and 200r/min for 16h to induce enzyme production.

Centrifuging at 4deg.C and 10000 Xg for 10min, discarding supernatant, collecting to obtain thallus, re-suspending thallus with 10mL of 0.2M Phosphate Buffer Solution (PBS) with pH of 7.5, performing ice bath ultrasonic wall breaking, and performing ultrasonic treatment for 60w, ultrasonic treatment for 10s, stopping for 5s, and operating for 30 min. Centrifuging at 4deg.C and 8000 Xg for 10min, and retaining supernatant to obtain crude enzyme solution. And (3) verifying whether the target protein is expressed or not through SDS-PAGE electrophoresis. Protein bands were visualized by coomassie blue staining, and markers were run under the same conditions as the samples for reference. SDS-PAGE gel electrophoresis is shown in FIG. 6. The molecular weight of the target protein calculated according to the amino acid sequence SEQ ID NO.1 is 59.5kda, and a significant protein accumulation around 63kda can be seen from the figure, which indicates that recombinant protease is obtained by successful induction of expression.

EXAMPLE 2 construction of M30011CPY mutant

The 271Tyr, 464Ile and 517Met of the target protein were selected as mutation sites, the pCold TF-CPY plasmid in example 1 was used as a mutation template, and two rounds of PCR site-directed mutagenesis was performed by overlap extension PCR using the sequences shown in Table 3 as primers.

TABLE 3 overlap extension PCR primers

The first round of PCR was performed with the constructed pCold TF-CPY plasmid as a template and pCold TF-F, Y271G/I464G/M517R-R and Y271G/I464G/M517R-F as primers to obtain the front and rear sequences containing the mutation sites, respectively, as shown in FIG. 7. The second round of PCR uses the front-stage and rear-stage products obtained by the first round of PCR as templates and pCold TF-F and pCold TF-R as primers to carry out PCR so as to obtain the complete sequence of the mutant. After the PCR is completed, the PCR products are subjected to 1% agarose gel electrophoresis detection and gel cutting recovery.

Recombinant plasmids pCold TF-CPY-Y271R, pCold TF-CPY-I464G and pCold TF-CPY-M517R were constructed, respectively, according to the method of example 1, and then BL21 (DE 3) was transformed by heat shock, the positive transformants induced enzyme production, and the SDS-PAGE gel electrophoresis of the crude enzyme solution was shown in FIG. 8, from which it was seen that there was a significant protein accumulation around 60 kda. The result shows that the recombinant protease is obtained by successful induction expression, and the amino acid sequence of the mutant is shown as SEQ ID NO. 2-4.

Purifying the crude enzyme solution with Ni column, eluting with equilibration buffer solution to obtain A ₂₈₀ <Eluting the hetero protein with 0.05, and then eluting and collecting the hetero protein with imidazole eluents with different concentrations of 50mM, 100mM, 200mM and 300mM in sequence. Samples eluted at each imidazole concentration were subjected to SDS-PAGE to finally select the imidazole content in the eluate for eluting the hybrid protein and the single protein of interest. In a wild-type purification pre-experiment, selectionThe results of SDS-PAGE of the eluted samples at the respective imidazole concentrations are shown in FIG. 9. Most of the proteins are eluted by using an equilibrium buffer solution, and the target proteins are obtained by using the concentration of 200mM imidazole, so that protein bands are accumulated most obviously, and the proteins are basically eluted. The target protein was eluted at a concentration of 200mM imidazole for the purification of 3 mutants, and SDS-PAGE thereof was shown in FIG. 10. The collected solution was stored at 4℃for further use.

EXAMPLE 3M 30011CPY and specific cleavage of aromatic amino acids by its mutant form

Polypeptides of different lengths: tripeptide (N)cbzGly-Gly-Phe), tripeptide (N-cbz-Gly-Phe-Phe) and pentapeptide (Phe-Gly-Leu-Gly-Phe) are respectively used as substrates, the enzyme activity of the CPY protein and 3 mutants for preparing the high F value oligopeptide by specifically cutting the aromatic amino acid is measured, and the content of free phenylalanine in the sample is measured by using a fluorescent high performance liquid chromatography. The unit of enzyme activity is defined as the amount of enzyme required (mg) to produce 1 μg of terminal amino acid per minute under the reaction conditions.

A500. Mu.L reaction system was constructed, and 30. Mu.L of the polypeptide substrate (1 mg/mL) was added to 5. Mu.g of purified enzyme, followed by pH7.5, and 0.2M PBS buffer was added to the mixture, followed by reaction in a water bath at 40℃for 20 minutes, followed by boiling for 5 minutes. The control was treated in the same manner with PBS buffer (pH 7.5, 0.2M) instead of the crude enzyme solution. And (3) respectively treating the reacted reaction liquid by using an OPA pre-column derivatization method to carry out fluorescence high performance liquid analysis.

TABLE 4M 30011CPY and mutant enzyme activities (U/mg) for various polypeptides

Table 4 shows that the enzyme activities of the primordial enzyme and the mutant for peptide substrates of different lengths, the activity of mutant enzyme M517R for each peptide substrate was significantly higher than that of the other mutant zymoenzymes. For N-cbz-Gly-Gly-Phe and Phe-Gly-Leu-Gly-Phe, mutants Y271R and I464G were significantly more active than the primordial enzyme. Prozymes are directed against tripeptide substrates N-cbzActivity of Gly-Phe-Phe compared to tripeptide substrate N-cbz-Gly-Gly-Phe has high activity.

EXAMPLE 4 preparation of chymotrypsin mutant enzyme P308R

Synthesizing a target gene CHY1 by a chemical synthesis method, wherein the nucleotide sequence is shown as SEQ ID NO. 18, analyzing restriction endonuclease recognition sites on multiple cloning sites of the target gene and a vector plasmid pCold TF, selecting two enzyme cutting sites NdeI and HindIII, designing primers to introduce the two enzyme cutting sites, and amplifying the target gene by PCR, wherein the primer sequences are CHY1-F and CHY1-R respectively as shown in Table 5. The amplified gene and plasmid vector pCold TF were digested with restriction enzymes NdeI and HindIII, respectively, and digested at 37℃for 3 hours, followed by detection by 1% (m/v) agarose gel electrophoresis and recovery of the digested CHY1 and linearized pCold TF vector plasmids with cohesive ends by gel cutting. The digested CHY1 and pCold TF plasmid are placed in a reaction system according to a molar ratio of 7:1, and are reacted overnight at 16 ℃ by using T4 DNA ligase to obtain recombinant plasmid pCold TF-CHY1.

To improve the specificity of che for aromatic amino acids, site-directed mutagenesis was performed on proline at position 308. The pCold TF-CHY1 recombinant plasmid was amplified by overlap extension PCR, and two rounds of amplification were performed using the sequences shown in Table 5 as primers.

TABLE 5 mutant design of upstream and downstream mutation primers

The resulting product was detected by 1% (m/v) agarose gel electrophoresis and cut for recovery. And (3) respectively carrying out double digestion on the pCold TF and the mutant sequence, and then adopting T4 DNA ligase to link the double digested mutant sequence and the pCold TF, thereby obtaining a recombinant plasmid pCold TF-CHY1-P308R. The recombinant plasmid was introduced into BL21 (DE 3) competent cells, positive transformants were selected on ampicillin resistance plates and verified by colony PCR followed by sequencing. And (5) performing IPTG induction on the bacterial liquid with correct verification to produce enzyme. Then breaking the wall by ultrasonic cell disruption to obtain crude enzyme solution. The recombinant protease is obtained by resuspension of thalli with a Phosphate Buffer Solution (PBS) of 10mL 0.2M,pH7.5 and verification of target protein by SDS-PAGE electrophoresis, and the recombinant protease is chymotrypsin mutant enzyme P308R, the amino acid sequence of which is shown as SEQ ID NO. 23, and the 308 site is mutated into arginine. Purifying the crude enzyme solution by Ni column to obtain chymotrypsin mutant enzyme P308R pure enzyme solution, wherein the activity of the chymotrypsin mutant enzyme P is 50.67U/mg for small molecule substrates (Leu-Gly-Tyr-Gly-Leu), and preserving at 4 ℃ for standby.

Application example 1 preparation of high F value oligopeptide by using rice bran protein as substrate

1. Preparation of rice bran protein

Adding n-hexane at a rice bran feed liquid ratio of 1:10, stirring at room temperature for degreasing for 3h, centrifuging (4000 Xg, 15 min) to remove n-hexane, adding n-hexane for degreasing again, and air-drying in a fume hood for 12h. Weighing 5g of defatted rice bran powder, adding 0.1mol/L NaOH solution according to a ratio of 1:19.6, stirring uniformly, placing in a constant-temperature shaking table at 40 ℃, oscillating and extracting rice bran protein for 4.7h, centrifuging (4000 Xg, 15 min), collecting supernatant, adding NaOH solution into precipitate, repeating the steps for extraction again, combining the obtained supernatant (recording volume) twice, regulating pH to 5.4 with 0.01mol/L HCl solution, centrifuging (4000 Xg, 15 min), discarding the supernatant, lightly washing 3 times with distilled water, collecting precipitate, and re-dissolving with 10mL of 0.2M pH=7.5 solution to obtain rice bran protein liquid.

2. Combined enzymolysis of different endopeptidases and exopeptidases

Endopeptidase: non-specific alkaline protease AP; the specific chymotrypsin mutant enzyme P308R enzyme prepared in example 4;

exopeptidase: nonspecific papain P and flavor protease FP; specific serine carboxypeptidase CPY prepared in example 2 and mutant enzyme Y271R, I464G, M517R.

The alkaline protease (enzyme activity: 200U/mg), papain (enzyme activity: 800U/mg) and flavourzyme (enzyme activity: 20U/mg) used were all purchased from Shanghai Seiyaku Biotechnology Co.

The combination of different endopeptidases and exopeptidases and the enzymatic conditions are as follows:

(1) AP group: treating the rice bran protein liquid obtained in the step (1) with alkaline protease AP, wherein enzymolysis conditions are as follows: pH=9, 50 ℃, and enzyme amount 1% (E/S, w/v) is added for enzymolysis for 4 hours;

(2) Ap+p group: the first step is endopeptidase enzymolysis with AP group; the second step of exopeptidase enzymolysis is to adjust the pH value of the enzymolysis liquid in the first step to be 7.0 and 55 ℃, add papain P and add 1% (E/S, w/v) of enzyme amount, and carry out enzymolysis for 4 hours;

(3) Ap+fp group: the first step is endopeptidase enzymolysis with AP group; and the second step of exopeptidase enzymolysis is to adjust the pH value of the enzymolysis liquid in the first step to 7.0, add the flavourzyme FP with the activity of 20U/mg according to the enzyme adding amount of 1%, hydrolyze for 4 hours under the water bath condition of 50 ℃, boil for 5 minutes and inactivate.

(4) P308r+fp group: the first step of endopeptidase enzymolysis is to treat the rice bran protein solution obtained in the step (1) with chymotrypsin mutant enzyme P308R under the following enzymolysis conditions: pH=7.5, 50 ℃, adding 1% (E/S, w/v) of enzyme, performing enzymolysis for 4 hours, and boiling the enzymolysis liquid for 5 minutes; the second step of exopeptidase enzymolysis is to adjust the pH value of the enzymolysis liquid in the first step to be 7.0, 50 ℃, add flavourzyme FP and enzyme quantity to be 1% (E/S, w/v) for 4 hours;

(5) P308r+p group: the first step of endopeptidase enzymolysis and P308R+FP group first step; the second step of exopeptidase enzymolysis with AP+P group;

(6) P308r+cpy group: the first step of endopeptidase enzymolysis and P308R+FP group first step; the second step of exopeptidase enzymolysis is to adjust the pH value of the enzymolysis liquid in the first step to 7.5, add the purified enzyme liquid according to the enzyme adding amount (E/S, w/v) of 1%, and carry out enzymolysis for 4 hours under the water bath condition of 40 ℃;

(7) Group P308 r+y271R: except for the second step, the peptidase is hydrolyzed by using mutant enzyme Y271R, and other steps are the same as those of the P308R+CPY group;

(8) Group P308 r+i464G: except for the second step, the peptidase is enzymatically hydrolyzed with mutant enzyme I464G, and the other steps are the same as those of the P308R+CPY group;

(9) Group P308 r+m517R: except for the second step, the peptidase is hydrolyzed by using mutant enzyme M517R, and other steps are the same as those of the P308R+CPY group;

stirring is continued during the enzymolysis, and the pH change is monitored and regulated by 1mol/L NaOH. After the reaction is finished, the reaction system is immediately placed in a boiling water bath and kept for 5min, and the reaction is terminated.

3. Determination of F value of enzymatic hydrolysate

And (3) regulating the pH value of the whole enzymolysis liquid to 4.5, adding active carbon according to the solid-to-liquid ratio of 1:10, adsorbing for 12 hours at 25 ℃, and filtering to remove active carbon. Ultrafiltration treatment was performed using a 3 kDa-sized ultrafiltration membrane to remove large molecular weight peptide chains, resulting in a high F-number oligopeptide solution.

The amino acid content of each sample was measured using high performance liquid chromatography. Each sample was hydrolyzed with 6M HCl at 110 ℃ for 22h prior to assay. The F value of each sample is defined as the molar ratio of branched amino acids to aromatic amino acids. FIG. 11 is a bar graph showing the F values of oligopeptide product solutions obtained from rice bran proteins treated with various combinations of enzymes. As can be seen from the experimental results, hydrolysis with non-specific alkaline protease AP alone gave an oligopeptide with an F value of 13.1. The F value of the sample prepared by using the CPY+P308R combination derived from the Aspergillus oryzae strain M30011 is 29.84, the F values of the oligopeptides prepared by using the specific mutants Y271R, I464G, M517R and P308R combination are 42.51, 38.20 and 43.16 respectively, the F values are respectively improved by 42.46%, 28.02% and 44.64% compared with the original enzyme, and the F values are increased by 3.29 times compared with the single enzyme, so that the high F value level is achieved. Meanwhile, the F value is better than the combination of nonspecific exopeptidase (flavourzyme FP and papain P) and specific endopeptidase P308R, and is more obviously better than the combination of nonspecific double enzymes (alkaline protease AP and papain P, alkaline protease AP and flavourzyme FP).

Sequence listing

<110> university of Beijing Industrial and commercial university

<120> a specific carboxypeptidase derived from Aspergillus oryzae for the preparation of high F-number oligopeptides

<160> 23

<170> SIPOSequenceListing 1.0

<210> 1

<211> 531

<212> PRT

<213> Aspergillus oryzae (Aspergillus oryzae)

<400> 1

Gly Arg Ala Ala His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly

1 5 10 15

Arg Gly Ser Met Ala Val Pro Pro Leu Gln Gln Val Leu Gly Arg Pro

20 25 30

Glu Glu Gly Met Ser Phe Ser Lys Pro Leu His Ala Phe Gln Glu Gln

35 40 45

Leu Lys Thr Leu Ser Glu Asp Ala Arg Lys Leu Trp Asp Glu Val Ala

50 55 60

Asn Tyr Phe Pro Asp Ser Met Asp His Ser Pro Ile Phe Ser Leu Pro

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Lys Lys His Thr Arg Arg Pro Asp Ser His Trp Asp His Ile Val Arg

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

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

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

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Thr Gly Tyr Leu Asp Asp Asn Gly Asn Asp Lys His Leu Phe Tyr Trp

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

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Leu Asn Gly Gly Pro Gly Cys Ser Ser Leu Thr Gly Leu Phe Met Glu

180 185 190

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

195 200 205

Phe Ser Trp Asn Ser Asn Ala Ser Val Ile Phe Leu Asp Gln Pro Val

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Asn Val Gly Tyr Ser Tyr Ser Gly Ser Ala Val Ser Asp Thr Val Ala

225 230 235 240

Ala Gly Lys Asp Val Tyr Ala Leu Leu Ser Leu Phe Phe Lys Gln Phe

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Pro Glu Tyr Ala Glu Gln Asp Phe His Ile Ala Gly Glu Ser Tyr Ala

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Gly His Tyr Ile Pro Val Phe Ala Ser Glu Ile Leu Ala His Lys Asn

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Arg Asn Ile Asn Leu Lys Ser Val Leu Ile Gly Asn Gly Leu Thr Asp

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Gly Leu Thr Gln Tyr Gly Tyr Tyr Arg Pro Met Gly Cys Gly Glu Gly

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

325 330 335

Ala Leu Pro Arg Cys Arg Ser Met Ile Glu Ser Cys Tyr Asn Ser Glu

340 345 350

Ser Ala Trp Val Cys Val Pro Ala Ser Ile Tyr Cys Asn Asn Ala Leu

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Ile Gly Pro Tyr Gln Arg Thr Gly Gln Asn Val Tyr Asp Val Arg Ser

370 375 380

Lys Cys Glu Asp Glu Ser Asn Leu Cys Tyr Lys Gly Met Gly Tyr Val

385 390 395 400

Ser Glu Tyr Leu Asn Lys Ala Glu Val Arg Glu Ala Val Gly Ala Glu

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Val Gly Gly Tyr Asp Ser Cys Asn Phe Asp Ile Asn Arg Asn Phe Leu

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Phe His Gly Asp Trp Met Lys Pro Tyr His Arg Leu Val Pro Gly Leu

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Leu Glu Gln Ile Pro Val Leu Ile Tyr Ala Gly Asp Ala Asp Tyr Ile

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Cys Asn Trp Leu Gly Asn Lys Ala Trp Thr Glu Ala Leu Glu Trp Pro

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

485 490 495

Gln Asn Glu His Thr Gly Lys Lys Ile Gly Gln Val Lys Ser His Gly

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

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Leu Pro Cys

530

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<211> 531

<212> PRT

<213> Aspergillus oryzae (Aspergillus oryzae)

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Gly Arg Ala Ala His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly

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Arg Gly Ser Met Ala Val Pro Pro Leu Gln Gln Val Leu Gly Arg Pro

20 25 30

Glu Glu Gly Met Ser Phe Ser Lys Pro Leu His Ala Phe Gln Glu Gln

35 40 45

Leu Lys Thr Leu Ser Glu Asp Ala Arg Lys Leu Trp Asp Glu Val Ala

50 55 60

Asn Tyr Phe Pro Asp Ser Met Asp His Ser Pro Ile Phe Ser Leu Pro

65 70 75 80

Lys Lys His Thr Arg Arg Pro Asp Ser His Trp Asp His Ile Val Arg

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Thr Gly Tyr Leu Asp Asp Asn Gly Asn Asp Lys His Leu Phe Tyr Trp

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

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Leu Asn Gly Gly Pro Gly Cys Ser Ser Leu Thr Gly Leu Phe Met Glu

180 185 190

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

195 200 205

Phe Ser Trp Asn Ser Asn Ala Ser Val Ile Phe Leu Asp Gln Pro Val

210 215 220

Asn Val Gly Tyr Ser Tyr Ser Gly Ser Ala Val Ser Asp Thr Val Ala

225 230 235 240

Ala Gly Lys Asp Val Tyr Ala Leu Leu Ser Leu Phe Phe Lys Gln Phe

245 250 255

Pro Glu Tyr Ala Glu Gln Asp Phe His Ile Ala Gly Glu Ser Arg Ala

260 265 270

Gly His Tyr Ile Pro Val Phe Ala Ser Glu Ile Leu Ala His Lys Asn

275 280 285

Arg Asn Ile Asn Leu Lys Ser Val Leu Ile Gly Asn Gly Leu Thr Asp

290 295 300

Gly Leu Thr Gln Tyr Gly Tyr Tyr Arg Pro Met Gly Cys Gly Glu Gly

305 310 315 320

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

325 330 335

Ala Leu Pro Arg Cys Arg Ser Met Ile Glu Ser Cys Tyr Asn Ser Glu

340 345 350

Ser Ala Trp Val Cys Val Pro Ala Ser Ile Tyr Cys Asn Asn Ala Leu

355 360 365

Ile Gly Pro Tyr Gln Arg Thr Gly Gln Asn Val Tyr Asp Val Arg Ser

370 375 380

Lys Cys Glu Asp Glu Ser Asn Leu Cys Tyr Lys Gly Met Gly Tyr Val

385 390 395 400

Ser Glu Tyr Leu Asn Lys Ala Glu Val Arg Glu Ala Val Gly Ala Glu

405 410 415

Val Gly Gly Tyr Asp Ser Cys Asn Phe Asp Ile Asn Arg Asn Phe Leu

420 425 430

Phe His Gly Asp Trp Met Lys Pro Tyr His Arg Leu Val Pro Gly Leu

435 440 445

Leu Glu Gln Ile Pro Val Leu Ile Tyr Ala Gly Asp Ala Asp Tyr Ile

450 455 460

Cys Asn Trp Leu Gly Asn Lys Ala Trp Thr Glu Ala Leu Glu Trp Pro

465 470 475 480

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

485 490 495

Gln Asn Glu His Thr Gly Lys Lys Ile Gly Gln Val Lys Ser His Gly

500 505 510

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

515 520 525

Leu Pro Cys

530

<210> 3

<211> 531

<212> PRT

<213> Aspergillus oryzae (Aspergillus oryzae)

<400> 3

Gly Arg Ala Ala His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly

1 5 10 15

Arg Gly Ser Met Ala Val Pro Pro Leu Gln Gln Val Leu Gly Arg Pro

20 25 30

Glu Glu Gly Met Ser Phe Ser Lys Pro Leu His Ala Phe Gln Glu Gln

35 40 45

Leu Lys Thr Leu Ser Glu Asp Ala Arg Lys Leu Trp Asp Glu Val Ala

50 55 60

Asn Tyr Phe Pro Asp Ser Met Asp His Ser Pro Ile Phe Ser Leu Pro

65 70 75 80

Lys Lys His Thr Arg Arg Pro Asp Ser His Trp Asp His Ile Val Arg

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Thr Gly Tyr Leu Asp Asp Asn Gly Asn Asp Lys His Leu Phe Tyr Trp

145 150 155 160

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

165 170 175

Leu Asn Gly Gly Pro Gly Cys Ser Ser Leu Thr Gly Leu Phe Met Glu

180 185 190

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

195 200 205

Phe Ser Trp Asn Ser Asn Ala Ser Val Ile Phe Leu Asp Gln Pro Val

210 215 220

Asn Val Gly Tyr Ser Tyr Ser Gly Ser Ala Val Ser Asp Thr Val Ala

225 230 235 240

Ala Gly Lys Asp Val Tyr Ala Leu Leu Ser Leu Phe Phe Lys Gln Phe

245 250 255

Pro Glu Tyr Ala Glu Gln Asp Phe His Ile Ala Gly Glu Ser Tyr Ala

260 265 270

Gly His Tyr Ile Pro Val Phe Ala Ser Glu Ile Leu Ala His Lys Asn

275 280 285

Arg Asn Ile Asn Leu Lys Ser Val Leu Ile Gly Asn Gly Leu Thr Asp

290 295 300

Gly Leu Thr Gln Tyr Gly Tyr Tyr Arg Pro Met Gly Cys Gly Glu Gly

305 310 315 320

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

325 330 335

Ala Leu Pro Arg Cys Arg Ser Met Ile Glu Ser Cys Tyr Asn Ser Glu

340 345 350

Ser Ala Trp Val Cys Val Pro Ala Ser Ile Tyr Cys Asn Asn Ala Leu

355 360 365

Ile Gly Pro Tyr Gln Arg Thr Gly Gln Asn Val Tyr Asp Val Arg Ser

370 375 380

Lys Cys Glu Asp Glu Ser Asn Leu Cys Tyr Lys Gly Met Gly Tyr Val

385 390 395 400

Ser Glu Tyr Leu Asn Lys Ala Glu Val Arg Glu Ala Val Gly Ala Glu

405 410 415

Val Gly Gly Tyr Asp Ser Cys Asn Phe Asp Ile Asn Arg Asn Phe Leu

420 425 430

Phe His Gly Asp Trp Met Lys Pro Tyr His Arg Leu Val Pro Gly Leu

435 440 445

Leu Glu Gln Ile Pro Val Leu Ile Tyr Ala Gly Asp Ala Asp Tyr Gly

450 455 460

Cys Asn Trp Leu Gly Asn Lys Ala Trp Thr Glu Ala Leu Glu Trp Pro

465 470 475 480

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

485 490 495

Gln Asn Glu His Thr Gly Lys Lys Ile Gly Gln Val Lys Ser His Gly

500 505 510

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

515 520 525

Leu Pro Cys

530

<210> 4

<211> 531

<212> PRT

<213> Aspergillus oryzae (Aspergillus oryzae)

<400> 4

Gly Arg Ala Ala His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly

1 5 10 15

Arg Gly Ser Met Ala Val Pro Pro Leu Gln Gln Val Leu Gly Arg Pro

20 25 30

Glu Glu Gly Met Ser Phe Ser Lys Pro Leu His Ala Phe Gln Glu Gln

35 40 45

Leu Lys Thr Leu Ser Glu Asp Ala Arg Lys Leu Trp Asp Glu Val Ala

50 55 60

Asn Tyr Phe Pro Asp Ser Met Asp His Ser Pro Ile Phe Ser Leu Pro

65 70 75 80

Lys Lys His Thr Arg Arg Pro Asp Ser His Trp Asp His Ile Val Arg

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Thr Gly Tyr Leu Asp Asp Asn Gly Asn Asp Lys His Leu Phe Tyr Trp

145 150 155 160

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

165 170 175

Leu Asn Gly Gly Pro Gly Cys Ser Ser Leu Thr Gly Leu Phe Met Glu

180 185 190

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

195 200 205

Phe Ser Trp Asn Ser Asn Ala Ser Val Ile Phe Leu Asp Gln Pro Val

210 215 220

Asn Val Gly Tyr Ser Tyr Ser Gly Ser Ala Val Ser Asp Thr Val Ala

225 230 235 240

Ala Gly Lys Asp Val Tyr Ala Leu Leu Ser Leu Phe Phe Lys Gln Phe

245 250 255

Pro Glu Tyr Ala Glu Gln Asp Phe His Ile Ala Gly Glu Ser Tyr Ala

260 265 270

Gly His Tyr Ile Pro Val Phe Ala Ser Glu Ile Leu Ala His Lys Asn

275 280 285

Arg Asn Ile Asn Leu Lys Ser Val Leu Ile Gly Asn Gly Leu Thr Asp

290 295 300

Gly Leu Thr Gln Tyr Gly Tyr Tyr Arg Pro Met Gly Cys Gly Glu Gly

305 310 315 320

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

325 330 335

Ala Leu Pro Arg Cys Arg Ser Met Ile Glu Ser Cys Tyr Asn Ser Glu

340 345 350

Ser Ala Trp Val Cys Val Pro Ala Ser Ile Tyr Cys Asn Asn Ala Leu

355 360 365

Ile Gly Pro Tyr Gln Arg Thr Gly Gln Asn Val Tyr Asp Val Arg Ser

370 375 380

Lys Cys Glu Asp Glu Ser Asn Leu Cys Tyr Lys Gly Met Gly Tyr Val

385 390 395 400

Ser Glu Tyr Leu Asn Lys Ala Glu Val Arg Glu Ala Val Gly Ala Glu

405 410 415

Val Gly Gly Tyr Asp Ser Cys Asn Phe Asp Ile Asn Arg Asn Phe Leu

420 425 430

Phe His Gly Asp Trp Met Lys Pro Tyr His Arg Leu Val Pro Gly Leu

435 440 445

Leu Glu Gln Ile Pro Val Leu Ile Tyr Ala Gly Asp Ala Asp Tyr Ile

450 455 460

Cys Asn Trp Leu Gly Asn Lys Ala Trp Thr Glu Ala Leu Glu Trp Pro

465 470 475 480

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

485 490 495

Gln Asn Glu His Thr Gly Lys Lys Ile Gly Gln Val Lys Ser His Gly

500 505 510

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

515 520 525

Leu Pro Cys

530

<210> 5

<211> 42

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

accctcgagg gatccgaatt ctcatcatca tcacagcagc gg 42

<210> 6

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

agcagagatt acctatctag accagactag cctcgggctg 40

<210> 7

<211> 1594

<212> DNA

<213> Aspergillus oryzae (Aspergillus oryzae)

<400> 7

gggcgcgcgg cgcatatggc tagcatgact ggtggacagc aaatgggtcg cggatccatg 60

gccgttccac ctcttcagca ggtgctgggt cgacctgagg agggcatgag cttctccaag 120

ccactccatg ccttccaaga gcagctcaag actctttcag aggatgctcg taaactctgg 180

gatgaagttg ccaactactt cccggacagc atggaccaca gccccatctt ctctcttccc 240

aagaagcaca cccgtcgtcc ggattctcat tgggaccaca ttgtccgtgg ctctgatgtc 300

cagaagatct gggtgaacaa tgccgacgga gagaaggaga gagagattga tggaaagctt 360

gaggcttacg atctcagggt caagaaagct gacccgagcg ctctcggtat cgacccgaat 420

gtgaagcaat acactggtta tctcgacgat aacggtaatg acaagcatct gttctactgg 480

ttcttcgaat ctcgcaatga tcctaagaac gaccccgtcg tgctctggct gaatggtggc 540

cccggttgct cttccctcac tggtctattt atggagctgg gacctagcag catcgatgag 600

aacattaagc ccgtctacaa tgacttctcg tggaactcta acgcctccgt cattttcctt 660

gatcagcccg tgaatgtcgg ctattcctac agtggctccg ctgttagcga cactgttgct 720

gctggcaagg atgtctacgc tctgctctct cttttcttca aacaattccc cgaatatgcg 780

gaacaggact tccatattgc cggcgagtcc tacgccgggc attacattcc agtctttgca 840

tccgagatct tggctcacaa gaaccgcaac atcaacctca agtcagtcct gatcggcaac 900

ggtctcacgg acggcctcac tcagtacggg tactaccgcc ccatgggatg tggcgagggt 960

ggctacaagg ctgtcctcga tgaggctacc tgcgaatcta tggacaatgc cctaccccgc 1020

tgtcggtcca tgatcgagtc ttgctataat tctgagagcg cctgggtctg tgtccctgcc 1080

tcgatttact gcaataatgc tcttatcgga ccttaccagc gcaccggaca gaacgtctac 1140

gatgttcgta gcaagtgtga ggatgaaagc aacctgtgct acaagggtat gggttacgtg 1200

agtgaatatc tcaacaaggc tgaggttcgc gaggccgtcg gagccgaagt tggtggttat 1260

gactcctgca acttcgacat caaccgtaac ttcctcttcc acggtgattg gatgaagccc 1320

taccaccgtc ttgttcccgg ccttctcgag cagatccctg tgctgatcta cgccggtgat 1380

gctgactaca tctgcaactg gctcggcaac aaggcctgga ccgaggctct tgaatggcct 1440

ggccagaagg agtacgcctc ggccgaactg gaggacctta agatcgagca gaacgaacac 1500

accggcaaga agatcggtca ggttaagtct catggaaatt tcaccttcat gcgtctctcc 1560

ggggagtcac gcgcaatcag actgctacca tgtg 1594

<210> 8

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

ccggaattct actaccagca gcggcctg 28

<210> 9

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gctctagacc agactagcct cgggctggt 29

<210> 10

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

attgccggcg agtcccgcgc cgggcattac att 33

<210> 11

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

aatgtaatgc ccggcgccgc gctcgccggc aat 33

<210> 12

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

ggtgatgctg actacggctg caactggctc ggc 33

<210> 13

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

gccgagccag ttgcagccgt agtcagcatc acc 33

<210> 14

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

ggaaatttca ccttccggcg tctctccggg gag 33

<210> 15

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

ctccccggag agacgccgga aggtgaaatt tcc 33

<210> 16

<211> 42

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

accctcgagg gatccgaatt ctcatcatca tcacagcagc gg 42

<210> 17

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

agcagagatt acctatctag accagactag cctcgggctg 40

<210> 18

<211> 1077

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

atgggcgcgc cgacccaggc ggcgagcctg catccgcaga ttctggaagc catgaaacgt 60

gatctgggcc tgaacgcgga acaggcgacc gttcgtgtgg cgcgtgaaat tcatgcgacc 120

gatgtgattg aacagctgcg tagcagcgtg gcgtttgcag gtgcgtggat tgatgcggat 180

gtgctgtata tcggcattac cgatcaggcg ctggccgatg aagttaccgc ggccggcgcg 240

accccgattg tgatgaccaa cagcctgagc aaactggaaa aagcgaaaga agatctggat 300

aaaatcttta ttggccgtgc gaacaccctg gaaaccagca gcgataccag cagcggcatt 360

gcgagctatt ttgtggatgt ggcggcgaac aaactggtga ttgaagccct ggccgatagc 420

catggccatg cggaacagct ggccgcgcag gttggtctga ccagcgaatt tgaagtgcgt 480

accgtggaaa ccatgccgac caccatggcg accgtgcagg gcggtgatgt gtattatatt 540

aaccgtagca gccgttgcag cattggcttt gcggtgacca ccggctttgt gagcgcgggt 600

cattgcggtg gtagcggtgc gagcgcgacc acctctagcg gtgaagcgct gggcaccttt 660

agcggcagcg tgtttccggg cagcgcggat atggcgtatg tgcgcaccgt gagcggtacg 720

gtgctgcgtg gctatattaa cggctatggc cagggcagct ttccggtgag cggcagcagc 780

gaagcggcgg tgggtgcgag catttgtcgt tctggcagca ccacccaggt gcattgcggc 840

accattggcg cgaaaggcgc gaccgtgaac tatccgcagg gcgcggttag cggtctgacc 900

cgtaccagcg tgtgtgcgga accgggcgat agcggtggca gcttttatag cggcagccag 960

gcgcagggtg ttacctctgg tggttctggc gattgtagcc gtggcggcac cacctatttt 1020

cagccggtga accgtattct gcagacctat ggcctgaccc tggtgaccgc gctcgag 1077

<210> 19

<211> 36

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

agcgtgtgtg cggaaggcgg cgatagcggt ggcagc 36

<210> 20

<211> 36

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

gctgccaccg ctatcgccgc cttccgcaca cacgct 36

<210> 21

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

taggatccat gggcgcgccg accca 25

<210> 22

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

taaagcttct cgagcgcggt cacca 25

<210> 23

<211> 359

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 23

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

1 5 10 15

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

20 25 30

Val Ala Arg Glu Ile His Ala Thr Asp Val Ile Glu Gln Leu Arg Ser

35 40 45

Ser Val Ala Phe Ala Gly Ala Trp Ile Asp Ala Asp Val Leu Tyr Ile

50 55 60

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

65 70 75 80

Thr Pro Ile Val Met Thr Asn Ser Leu Ser Lys Leu Glu Lys Ala Lys

85 90 95

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

100 105 110

Ser Ser Asp Thr Ser Ser Gly Ile Ala Ser Tyr Phe Val Asp Val Ala

115 120 125

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

130 135 140

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

145 150 155 160

Thr Val Glu Thr Met Pro Thr Thr Met Ala Thr Val Gln Gly Gly Asp

165 170 175

Val Tyr Tyr Ile Asn Arg Ser Ser Arg Cys Ser Ile Gly Phe Ala Val

180 185 190

Thr Thr Gly Phe Val Ser Ala Gly His Cys Gly Gly Ser Gly Ala Ser

195 200 205

Ala Thr Thr Ser Ser Gly Glu Ala Leu Gly Thr Phe Ser Gly Ser Val

210 215 220

Phe Pro Gly Ser Ala Asp Met Ala Tyr Val Arg Thr Val Ser Gly Thr

225 230 235 240

Val Leu Arg Gly Tyr Ile Asn Gly Tyr Gly Gln Gly Ser Phe Pro Val

245 250 255

Ser Gly Ser Ser Glu Ala Ala Val Gly Ala Ser Ile Cys Arg Ser Gly

260 265 270

Ser Thr Thr Gln Val His Cys Gly Thr Ile Gly Ala Lys Gly Ala Thr

275 280 285

Val Asn Tyr Pro Gln Gly Ala Val Ser Gly Leu Thr Arg Thr Ser Val

290 295 300

Cys Ala Glu Arg Gly Asp Ser Gly Gly Ser Phe Tyr Ser Gly Ser Gln

305 310 315 320

Ala Gln Gly Val Thr Ser Gly Gly Ser Gly Asp Cys Ser Arg Gly Gly

325 330 335

Thr Thr Tyr Phe Gln Pro Val Asn Arg Ile Leu Gln Thr Tyr Gly Leu

340 345 350

Thr Leu Val Thr Ala Leu Glu

355

Claims

1. A carboxypeptidase whose amino acid sequence is shown in SEQ ID NO.1 is derived from Aspergillus oryzaeAspergillus oryzae) M30011 with preservation number of CGMCC No.12371.

2. A carboxypeptidase has an amino acid sequence shown in any one of SEQ ID NOs 2-4.

3. A carboxypeptidase-encoding nucleotide according to claim 1 or 2.

4. A plasmid or engineering bacterium comprising the coding nucleotide of claim 3.

5. The engineering bacterium according to claim 4, wherein the engineering bacterium is escherichia coli.

6. Use of the engineered bacterium of claim 4 or 5 in the preparation of carboxypeptidase.

7. Use of a carboxypeptidase according to claim 1 or 2 for the preparation of a high F-number oligopeptide.