CN107400666B

CN107400666B - A kind of aminopeptidase and its encoding gene and application

Info

Publication number: CN107400666B
Application number: CN201710812644.4A
Authority: CN
Inventors: 叶茂; 邓毛程; 李静; 张远平; 陈维新; 刘慧平
Original assignee: Guangdong Light Industry Vocational And Technical College
Current assignee: Guangdong Light Industry Vocational And Technical College
Priority date: 2017-09-11
Filing date: 2017-09-11
Publication date: 2019-11-22
Anticipated expiration: 2037-09-11
Also published as: CN107400666A

Abstract

The invention discloses a kind of aminopeptidase and its encoding gene and applications.The amino acid sequence of the aminopeptidase is as shown in SEQ ID NO.1.The present invention also provides the nucleotide sequences for encoding the aminopeptidase.The aminopeptidase has the function of that almost all of amino acid residue can be hydrolyzed have good application potential, and to abundant aminopeptidase family member, develops China's traditional condiment environmental resource and be all of great significance.Therefore, it can be used for preparing flavor peptide, flavor peptide can be made by the steps: aminopeptidase provided by the invention is added in the hydrolysis reaction system containing protein substrate, 2~6h of enzymatic hydrolysis reaction is carried out under the conditions of 25~35 DEG C, obtains corresponding flavor peptide.

Description

A kind of aminopeptidase and its encoding gene and application

Technical field

The invention belongs to genetic engineering and enzyme engineering field, in particular to a kind of aminopeptidase and its encoding gene and application.

Background technique

Aminopeptidase is a kind of N-terminal sequential hydrolysis amino acid from polypeptide chain, the hydrolase for making amino acid separate out one by one, Its substrate-function range generally more wide spectrum, can not only hydrolyzed peptide, and complete protein molecule can be hydrolyzed.Aminopeptidase It is mainly used in protein hydrolysis, flavor peptides and biologically active polypeptide preparation and the fields such as biology and medicine: working as protein When macromolecular digests, the hydrophobic amino acid contained in peptide chain is exposed, and contacts taste bud and bitter taste is presented, and these hydrophobicitys Amino acid is normally at peptide chain end, and utilizes the further aminosal hydrolyzate of aminopeptidase, can remove its bitter taste；With egg White enzyme is used in combination, and in the preparation process of soy sauce brewing, fish sauce production and other proteolysis products, egg not only can be improved The utilization rate of white matter, and the delicious compounded amino acid hydrolysis liquid of unique flavor, flavour can be formed；Utilize the enzymatic hydrolysis system of aminopeptidase The active peptide of the standby characteristics such as anticancer, anti-oxidant, antibacterial；In terms of medical usage, leucine aminopeptidase etc. also acts as protein The molecular tool of sequencing, prolidase can effectively clear neurotoxicity chemical poison and organophosphorus pesticide, can not only be used for curing Antidote on, and can be used as environment disinfectant.Thus, aminopeptidase has very high researching value and broader practice Prospect.

Metagenomics (Metagenomics) are the fast developments recently as microbiology and modern life science The emerging science and technology risen.Its basic research thinking is the genomic DNA of all microorganisms in direct extraction environment, It is cloned into suitable carrier, constructs macro genomic library, the information nucleic acid of microorganisms whole in environment is collected together, and transports Useful enzyme, antibiotic, active material etc. are obtained from library with sequence screening or functional screening mode.It is this not depend on completely The technique extension of the separation and culture of microorganism microbiological Research Thinking and method in environment, to be given birth to naturally from different New genetic resources and active material are found in Anticipated transient without scram in border provides strong technological means.

Up to the present, the research that new aminopeptidase gene is found from macro genomic library, there is no relevant report both at home and abroad Road.

Summary of the invention

The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide a kind of aminopeptidase.The enzyme is to N- Terminal amino acid residue specificity is low, can hydrolyze almost all of amino acid residue, to abundant aminopeptidase family member, develops new Proteolysis is all of great significance at the approach of flavor peptides.

Another object of the present invention is to provide the genes for encoding the aminopeptidase.

A further object of the present invention is to provide the applications of the aminopeptidase.

The purpose of the invention is achieved by the following technical solution: a kind of aminopeptidase, amino acid sequence are as follows:

MNFQVQDAAIVIEAVIVALAFFAEKIIFAQQLDAAITGSLQALLAHKGISTVGYAVSLLHSLAGLNVD RYYVGLEGQQLAYGIQTLLRKAGDAFTTIKGGAVSDEQIKLPSFTKQRLDAAVLHIAVVSPGHGAYELTYSKKKDK QELFEFTAIALDDAQLDLEGFISVGDVTGVLLNFARQLGNAHPHVLTPAIKTAEEAVALFGKTDYDVKLDKEQMEG IGNGSLLAVKKGSENIPRLIVLTYNGKEDAAPVVALVGPGITFDSGGYSLKTRQGMEFMKFDMGCGAAVGGTVEAI GHLLPPKNIVGVIPASDNIVNGEAISPLDVGTSLSGYTIEVMNTFPEGRLLLADAVFYAYQDGPGVLADVATLTGA YIVVLGAHKTAGAMINPGILFNQLLDASEEVDEPAGLPAIWGTEGGVVTSDVADLPNYGGRDGTALFEGMFLTEEP ENTPWLYLDIAGGSRIEDAVLIGPDGAEGLLVPTLVDLLTREGEAEA。

A kind of nucleotide sequence is the nucleotide sequence of the coding aminopeptidase；Preferably intronless has The nucleotide sequence of the complete opening code-reading frame of 1485bp；Sequence more preferably as follows:

ATGAACTTCCAGGTTCAGGACGCTGCTATCGTTATCGAAGCTGTTATCGTTGCTCTGGCTTTCTTCGC TGAAAAAATCATCTTCGCTCAGCAGCTGGACGCTGCTATCACCGGTTCTCTGCAGGCTCTGCTGGCTCACAAAGGT ATCTCTACCGTTGGTTACGCTGTTTCTCTGCTGCACTCTCTGGCTGGTCTGAACGTTGACCGTTACTACGTTGGTC TGGAAGGTCAGCAGCTGGCTTACGGTATCCAGACCCTGCTGCGTAAAGCTGGTGACGCTTTCACCACCATCAAAGG TGGTGCTGTTTCTGACGAACAGATCAAACTGCCGTCTTTCACCAAACAGCGTCTGGACGCTGCTGTTCTGCACATC GCTGTTGTTTCTCCGGGTCACGGTGCTTACGAACTGACCTACTCTAAAAAAAAAGACAAACAGGAACTGTTCGAAT TCACCGCTATCGCTCTGGACGACGCTCAGCTGGACCTGGAAGGTTTCATCTCTGTTGGTGACGTTACCGGTGTTCT GCTGAACTTCGCTCGTCAGCTGGGTAACGCTCACCCGCACGTTCTGACCCCGGCTATCAAAACCGCTGAAGAAGCT GTTGCTCTGTTCGGTAAAACCGACTACGACGTTAAACTGGACAAAGAACAGATGGAAGGTATCGGTAACGGTTCTC TGCTGGCTGTTAAAAAAGGTTCTGAAAACATCCCGCGTCTGATCGTTCTGACCTACAACGGTAAAGAAGACGCTGC TCCGGTTGTTGCTCTGGTTGGTCCGGGTATCACCTTCGACTCTGGTGGTTACTCTCTGAAAACCCGTCAGGGTATG GAATTCATGAAATTCGACATGGGTTGCGGTGCTGCTGTTGGTGGTACCGTTGAAGCTATCGGTCACCTGCTGCCGC CGAAAAACATCGTTGGTGTTATCCCGGCTTCTGACAACATCGTTAACGGTGAAGCTATCTCTCCGCTGGACGTTGG TACCTCTCTGTCTGGTTACACCATCGAAGTTATGAACACCTTCCCGGAAGGTCGTCTGCTGCTGGCTGACGCTGTT TTCTACGCTTACCAGGACGGTCCGGGTGTTCTGGCTGACGTTGCTACCCTGACCGGTGCTTACATCGTTGTTCTGG GTGCTCACAAAACCGCTGGTGCTATGATCAACCCGGGTATCCTGTTCAACCAGCTGCTGGACGCTTCTGAAGAAGT TGACGAACCGGCTGGTCTGCCGGCTATCTGGGGTACCGAAGGTGGTGTTGTTACCTCTGACGTTGCTGACCTGCCG AACTACGGTGGTCGTGACGGTACCGCTCTGTTCGAAGGTATGTTCCTGACCGAAGAACCGGAAAACACCCCGTGGC TGTACCTGGACATCGCTGGTGGTTCTCGTATCGAAGACGCTGTTCTGATCGGTCCGGACGGTGCTGAAGGTCTGCT GGTTCCGACCCTGGTTGACCTGCTGACCCGTGAAGGTGAAGCTGAAGCT。

Above-mentioned nucleotide sequence can be obtained by chemical synthesis process, can also be made by the steps to obtain:

One, traditional fermented food environmental sample is extracted, total genome is extracted and purifies；

Two, total genome after purification is handled through Sau3AI digestion；

Three, digestion products are connected on pUC18/BamHI (BAP) carrier, are constructed in electroporated bacillus coli DH 5 alpha Traditional fermented food environment macro genomic library screens ammonia peptide with liquid selective medium and solid selection medium from library Enzyme positive clone, obtains positive clone molecule by high flux screening；

Four, compare design primer through sequencing and Blast, using the plasmid of positive clone molecule as template, with designed primer The reaction of chain type enzymatic polymerization is carried out, so that clone obtains above-mentioned nucleotide sequence.

Traditional fermented food described in step 1 includes soy sauce, fermented bean curd, beans sauce and sausage etc..

The composition of Selective agar medium described in step 3 is as follows: 100 μ g/ml ampicillins, the bright ammonia of 10mmol/L L- Without amino acid yeast nitrogen, solvent is water by acid -4- nitroaniline, 1 (w/v) % glucose and 0.5 (w/v) %.

Designed primer described in step 4 is as follows:

Upstream primer F1:5 '-CGCGGATCCATGAACTTCCAGGTTCAG-3 '；

Downstream primer F2:5 '-CGGAAGCTTAGCTTCACCTTCACGGTCA-3 '.

In addition, can be obtained by chemical synthesis process, or adopt the present invention also provides the preparation method of above-mentioned aminopeptidase It expresses to obtain with above-mentioned nucleotide sequence, include the following steps:

(1) nucleotide sequence is cloned into expression vector, then be transferred in expression cell, obtained containing the thin of recombinant vector Born of the same parents；

(2) cell containing recombinant vector obtained to step (1) is cultivated, and is induced through IPTG, separated from culture, Purifying obtains aminopeptidase.The aminopeptidase has the function of that almost all of amino acid residue can be hydrolyzed.

Expression vector described in step (1) is preferably pET-32a (+).

Expression cell described in step (1) is preferably e. coli bl21 (DE3).

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

Specific step is as follows for induction described in step (2): when the cell containing recombinant vector grows to OD₆₀₀=1.0 When be added IPTG to final concentration 0.8mM, 20 DEG C, cultivate 20h under 200r/min.

Isolated mode described in step (2) is preferably centrifuged.

In addition, the present invention also provides the aminopeptidases to prepare the application in flavor peptide, following steps are preferably included: The aminopeptidase is added in the hydrolysis reaction system containing protein substrate, it is anti-that enzyme hydrolysis is carried out under the conditions of 25~35 DEG C 2~6h is answered, corresponding flavor peptide is obtained.

The protein substrate includes soybean protein, zein, wheat gluten and Yeast protein etc..

The pH value of the hydrolysis reaction system is 6~8；Preferably 7.

Buffer solution in the hydrolysis reaction system is preferably phosphate buffer.

Mass concentration of the protein substrate in the hydrolysis reaction system is 1%~10%；Preferably 1%.

The present invention has the following advantages and effects with respect to the prior art:

(1) compared with the prior art, present invention application technique of metagenome is from China's traditional condiment environment macro genome The aminopeptidase that library obtains, it was found that the enzyme has the function of that almost all of amino acid residue can be hydrolyzed have good answer With potentiality, and to abundant aminopeptidase family member, develops China's traditional condiment environmental resource and be all of great significance.

(2) it originally returns and is studied by generating the optimum condition in flavor peptide in protein hydrolysate to the enzyme, it was found that It is generated in flavor peptide reaction by the hydrolysis of substrate raw material of albumen such as soybean protein, zein, wheat gluten, Yeast proteins, Aminopeptidase of the invention carries out enzymic catalytic reaction 2-6h under the conditions of 25-35 DEG C, and the mass concentration of the substrate is 1%~ 10%, generation flavor peptide, flavor peptide molecular weight≤3000Da, sense organ can efficiently, rapidly be hydrolyzed using above-mentioned reaction condition Identification has characteristic flavor on basis, has good application value.

Detailed description of the invention

Fig. 1 is the SDS-PAGE electrophoresis of aminopeptidase purifying provided by the invention；Wherein, swimming lane M is albumen Marker；Swimming Road 1 and 2 is respectively recombinant protein crude enzyme liquid (not purifying), and swimming lane 3 is albumen after purification.

Fig. 2 is the substrate specificity measurement result figure of the aminopeptidase of purifying.

Fig. 3 is the optimal reactive temperature measurement result figure of the aminopeptidase of purifying.

Fig. 4 is the optimal reaction pH value measurement result figure of the aminopeptidase of purifying.

Specific embodiment

Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited In this.

The acquisition of 1 aminopeptidase gene of embodiment and protein hydrolysate generate the verifying of flavor peptide function

1, the acquisition of aminopeptidase gene

From China's traditional fermented food --- sauce fermentation environmental sample (i.e. moromi) constructs fermented food environment macro gene Group library.With containing 100 μ g/ml ampicillins, 10mmol/L L-Leu -4- nitroaniline, 1 (w/v) % glucose and Culture medium of 0.5 (w/v) % without amino acid yeast nitrogen (YNB) alternatively culture medium.Cell in library is connect with photocopy Kind needle copies on solid selective medium plate, cultivates 3 days at 37 DEG C, and it is existing that screening periphery of bacterial colonies has yellow hydrolysis to iris out Positive clone molecule screens aminopeptidase positive colony from library.

The plasmid for extracting above-mentioned positive clone molecule is sent to sequencing company and is sequenced, and the nucleic acid sequence an of aminopeptidase is obtained Column, the sequence are named as an_105 as shown in SEQ ID NO.2 by 1485 base compositions, nucleic acid sequence；The nucleic acid The polypeptide of coding, contains 495 amino acid, and amino acid sequence is named as AN_105 as shown in SEQ ID NO.1.

2, the clone of genetic fragment

According to nucleic acid an_105 sequence, amplimer: upstream primer F1:5 '-is designed CGCGGATCCATGAACTTCCAGGTTCAG-3 ' (underscore is BamHI restriction enzyme site sequence)；Downstream primer F2:5 '- CGGAAGCTTAGCTTCACCTTCACGGTCA-3 ' (underscore is HindIII restriction enzyme site sequence).

Using the plasmid of positive clone molecule as template, using F1 and F2 as primer, PCR amplification, reaction system are carried out are as follows: PrimeSTAR^TM0.3 μ l of HS DNA Ploymerase (2.5U/ μ l), 5 × buffer 6 μ l, dNTP Mix (2.5mM) 2.4 μ l, 0.6 μ l of template (100ng/ μ l), primers F 1 and each 0.3 μ l of F2 (10mM), ultrapure water complement to 50 μ l.PCR reaction condition: 95 DEG C Initial denaturation 2min；98 DEG C of denaturation 10s, 68 DEG C of annealing 15s, 72 DEG C of extension 1.5min, 30 recycle；72 DEG C of extension 10min, 4 DEG C.

BamHI and HindIII double digestion 16h will be used after PCR product PCR product Purification Kit, it is same with passing through PET-32a (+) (Invitrogen) expression vector of sample processing is attached, electrotransformation to expressive host e. coli bl21 (DE3), by resistance screening, picking positive clone molecule extracts Plasmid DNA, carry out sequence verification find its nucleotide sequence with Sequence in SEQ ID NO.2 is identical, can effectively obtain target gene fragment.

3, the acquisition of Aminopeptidase A N_105 is recombinated

The strain inoculated through sequence verification correct plasmid to the LB Liquid Culture for containing 100 μ g/ml ampicillins will be contained In base, 30 DEG C, cultivate 14h under 250r/min, by the inoculum concentration of 2 (v/v) % be forwarded to 100ml containing 100 μ g/ml ammonia benzyl moulds In the LB liquid medium of element, when growing to OD₆₀₀Isopropylthio-β-D-galactoside is added when=1.0 to final concentration 0.8mM, 20 DEG C, 20h is cultivated under 200r/min, 12000r/min is centrifuged 5min, abandons supernatant, thallus is resuspended in 20ml 50mM In Tri-HCl (pH7.5), being crushed thallus with sonicator, (200w the time 10 seconds, is spaced 15 seconds, 5 minutes whole, ice Bath), 4 DEG C, 13000r/min is centrifuged 15min, collects supernatant, that is, obtains recombination aminopeptidase crude enzyme liquid.

The His label protein purification kit Proband of aminopeptidase crude enzyme liquid Invitrogen company will be recombinated Purification system carries out purification of recombinant proteins, and concrete operation step is carried out by the said firm's product description.After purification Recombinant protein liquid nitrogen it is quick-frozen, save into ultra low temperature freezer.The concentration of recombinant protein after purification is 290 μ g/ml.

Crude enzyme liquid and albumen after purification are measured by polyacrylamide gel electrophoresis, as a result as shown in Figure 1.As it can be seen that logical It crosses His label protein purification kit Proband Purification system and can succeed and purify to obtain this hair from crude enzyme liquid Bright aminopeptidase.

4, protein hydrolysate generates the verifying of flavor peptide function

By 20 μ l of above-mentioned recombination aminopeptidase crude enzyme liquid 2ml or the enzyme solution of purifying, 8ml pH7.0 phosphate buffer is added (0.2M biphosphate sodium water solution 39ml and 0.2M disodium hydrogen phosphate aqueous solution 61ml mixing) is used as reaction medium, tests respectively Different substrates (albumen such as soybean protein, zein, wheat gluten, Yeast protein), concentration are 1 (w/w) %, 30 DEG C of water-baths After reacting 6h, hydrolysis generates corresponding flavor peptide, flavor peptide molecular weight≤3000Da, and naked eyes evaluation has characteristic flavor on basis.Product is logical It crosses GC-MS and carries out Structural Identification.

Embodiment 2 recombinates the analysis of Aminopeptidase A N_105 substrate specificity

The 10 μ l of enzyme solution of purifying is added in 100mM Tris-HCl buffer (pH7.0), 1mM substrate, in 35 DEG C of measurement extinctions Value A_405nm.Measure substrate are as follows: L-Leu -4- nitroaniline (L-Leu-PNA, L-Leucine p-nitroanilide Hydrochloride, CAS 16010-98-3), L-Methionine -4- nitroaniline (L-Met-PNA, L-Methionine p- Nitroanilide, CAS 6042-04-2), L-PROLINE -4- nitroaniline (L-Pro-PNA, L-Proline p- Nitroanilide trifluoroacetate salt, CAS 108321-19-3), L-lysine -4- nitroaniline (L- Lys-PNA, L-Lysine p-nitroanilide dihydrobromide, CAS40492-96-4), L-arginine -4- nitro Aniline (L-Arg-PNA, L-Arginine p-nitroanilide dihydrochloride, CAS 40127-11-5), N- penta Two acyls-L-phenylalanine -4- nitroaniline (L-Phe-PNA, N-Glutaryl-L-phenylalanine p- Nitroanilide, CAS 5800-34-0), Pidolidone -4- nitroaniline (L-Glu-PNA, L-Glutamic acid γ - (p-nitroanilide) hydrochloride, CAS67953-08-6), D-phenylalanine-valine -4- nitroaniline (D- Phe-Val-PNA, D-Phe-Val-p-nitroanilide, CAS 108321-89-7), D-Val-leucine-lysine- 4- nitroaniline (D-Val-Leu-Lys-4-PNA, D-Val-Leu-Lys p-nitroanilide dihydrochloride), N- benzoyl-tyrosine -4- nitroaniline (N-Benzoyl-L-Tyr-PNA, N-Benzoyl-L-tyrosine p- nitroanilide,CAS 6154-45-6).As a result as shown in Fig. 2, recombination Aminopeptidase A N_105 has most substrate Catalytic activity, wherein the highest substrate of catalytic activity is L-Leu -4- nitroaniline (L-Leu-PNA), is secondly L- essence ammonia Acid -4- nitroaniline (L-Arg-PNA) and L-lysine -4- nitroaniline (L-Lys-PNA).

The measurement of the recombination Aminopeptidase A N_105 optimal reactive temperature of embodiment 3

Purifying is added using 1mM L-Leu -4- nitroaniline as substrate in 100mM Tris-HCl buffer (pH7.0) 10 μ l of enzyme solution afterwards, respectively in 20,25,30,35,40,45,50,55 and 60 DEG C of measurement light absorption value A_405nm.As a result as shown in figure 3, Aminopeptidase A N_105 is recombinated in 25~50 DEG C of catalytic activity still with higher, optimal reactive temperature is 35 DEG C.

The measurement of the recombination Aminopeptidase A N_105 optimal reaction pH value of embodiment 4

Using 1mM L-Leu -4- nitroaniline as substrate, 10 μ l of enzyme solution after purification is added, respectively in pH4.0~7.0 Under the different pH conditions such as (100mM phosphate buffer) and pH7.0~9.0 (Tris-HCl buffer), 35 DEG C of measurement extinctions Value A_405nm.As a result as shown in figure 4, recombination Aminopeptidase A N_105 optimal reaction pH is 7.0, still there is higher enzyme in pH4.0~9.0 Vigor.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

Sequence table

<110>Guangdong Industry Technical College

<120>a kind of aminopeptidase and its encoding gene and application

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 495

<212> PRT

<213>unknown (Unknown)

<400> 1

Met Asn Phe Gln Val Gln Asp Ala Ala Ile Val Ile Glu Ala Val Ile

1 5 10 15

Val Ala Leu Ala Phe Phe Ala Glu Lys Ile Ile Phe Ala Gln Gln Leu

20 25 30

Asp Ala Ala Ile Thr Gly Ser Leu Gln Ala Leu Leu Ala His Lys Gly

35 40 45

Ile Ser Thr Val Gly Tyr Ala Val Ser Leu Leu His Ser Leu Ala Gly

50 55 60

Leu Asn Val Asp Arg Tyr Tyr Val Gly Leu Glu Gly Gln Gln Leu Ala

65 70 75 80

Tyr Gly Ile Gln Thr Leu Leu Arg Lys Ala Gly Asp Ala Phe Thr Thr

85 90 95

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

100 105 110

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

115 120 125

Pro Gly His Gly Ala Tyr Glu Leu Thr Tyr Ser Lys Lys Lys Asp Lys

130 135 140

Gln Glu Leu Phe Glu Phe Thr Ala Ile Ala Leu Asp Asp Ala Gln Leu

145 150 155 160

Asp Leu Glu Gly Phe Ile Ser Val Gly Asp Val Thr Gly Val Leu Leu

165 170 175

Asn Phe Ala Arg Gln Leu Gly Asn Ala His Pro His Val Leu Thr Pro

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Arg Gln Gly Met Glu Phe Met Lys Phe Asp Met Gly Cys Gly Ala Ala

275 280 285

Val Gly Gly Thr Val Glu Ala Ile Gly His Leu Leu Pro Pro Lys Asn

290 295 300

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

305 310 315 320

Ile Ser Pro Leu Asp Val Gly Thr Ser Leu Ser Gly Tyr Thr Ile Glu

325 330 335

Val Met Asn Thr Phe Pro Glu Gly Arg Leu Leu Leu Ala Asp Ala Val

340 345 350

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

355 360 365

Leu Thr Gly Ala Tyr Ile Val Val Leu Gly Ala His Lys Thr Ala Gly

370 375 380

Ala Met Ile Asn Pro Gly Ile Leu Phe Asn Gln Leu Leu Asp Ala Ser

385 390 395 400

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

405 410 415

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

420 425 430

Arg Asp Gly Thr Ala Leu Phe Glu Gly Met Phe Leu Thr Glu Glu Pro

435 440 445

Glu Asn Thr Pro Trp Leu Tyr Leu Asp Ile Ala Gly Gly Ser Arg Ile

450 455 460

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

465 470 475 480

Pro Thr Leu Val Asp Leu Leu Thr Arg Glu Gly Glu Ala Glu Ala

485 490 495

<210> 2

<211> 1485

<212> DNA

<213>unknown (Unknown)

<400> 2

atgaacttcc aggttcagga cgctgctatc gttatcgaag ctgttatcgt tgctctggct 60

ttcttcgctg aaaaaatcat cttcgctcag cagctggacg ctgctatcac cggttctctg 120

caggctctgc tggctcacaa aggtatctct accgttggtt acgctgtttc tctgctgcac 180

tctctggctg gtctgaacgt tgaccgttac tacgttggtc tggaaggtca gcagctggct 240

tacggtatcc agaccctgct gcgtaaagct ggtgacgctt tcaccaccat caaaggtggt 300

gctgtttctg acgaacagat caaactgccg tctttcacca aacagcgtct ggacgctgct 360

gttctgcaca tcgctgttgt ttctccgggt cacggtgctt acgaactgac ctactctaaa 420

aaaaaagaca aacaggaact gttcgaattc accgctatcg ctctggacga cgctcagctg 480

gacctggaag gtttcatctc tgttggtgac gttaccggtg ttctgctgaa cttcgctcgt 540

cagctgggta acgctcaccc gcacgttctg accccggcta tcaaaaccgc tgaagaagct 600

gttgctctgt tcggtaaaac cgactacgac gttaaactgg acaaagaaca gatggaaggt 660

atcggtaacg gttctctgct ggctgttaaa aaaggttctg aaaacatccc gcgtctgatc 720

gttctgacct acaacggtaa agaagacgct gctccggttg ttgctctggt tggtccgggt 780

atcaccttcg actctggtgg ttactctctg aaaacccgtc agggtatgga attcatgaaa 840

ttcgacatgg gttgcggtgc tgctgttggt ggtaccgttg aagctatcgg tcacctgctg 900

ccgccgaaaa acatcgttgg tgttatcccg gcttctgaca acatcgttaa cggtgaagct 960

atctctccgc tggacgttgg tacctctctg tctggttaca ccatcgaagt tatgaacacc 1020

ttcccggaag gtcgtctgct gctggctgac gctgttttct acgcttacca ggacggtccg 1080

ggtgttctgg ctgacgttgc taccctgacc ggtgcttaca tcgttgttct gggtgctcac 1140

aaaaccgctg gtgctatgat caacccgggt atcctgttca accagctgct ggacgcttct 1200

gaagaagttg acgaaccggc tggtctgccg gctatctggg gtaccgaagg tggtgttgtt 1260

acctctgacg ttgctgacct gccgaactac ggtggtcgtg acggtaccgc tctgttcgaa 1320

ggtatgttcc tgaccgaaga accggaaaac accccgtggc tgtacctgga catcgctggt 1380

ggttctcgta tcgaagacgc tgttctgatc ggtccggacg gtgctgaagg tctgctggtt 1440

ccgaccctgg ttgacctgct gacccgtgaa ggtgaagctg aagct 1485

<210> 3

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

cgcggatcca tgaacttcca ggttcag 27

<210> 4

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

cggaagctta gcttcacctt cacggtca 28

Claims

1. a kind of aminopeptidase, it is characterised in that: its amino acid sequence is as shown in SEQ ID NO.1.

2. a kind of nucleotide sequence, it is characterised in that: be the nucleotide sequence for encoding aminopeptidase described in claim 1.

3. nucleotide sequence according to claim 2, it is characterised in that: the nucleotide sequence be for intronless, Sequence with the complete opening code-reading frame of 1485bp.

4. nucleotide sequence according to claim 3, it is characterised in that: the nucleotide sequence such as SEQ ID NO.2 It is shown.

5. the preparation method of aminopeptidase described in claim 1, it is characterised in that include the following steps:

(1) the described in any item nucleotide sequences of claim 2~4 are cloned into expression vector, then are transferred in expression cell, Obtain the cell containing recombinant vector；

(2) cell containing recombinant vector obtained to step (1) is cultivated, and is induced through IPTG, separates from culture, is pure Change, obtains aminopeptidase.

6. the preparation method of aminopeptidase according to claim 5, it is characterised in that:

Expression vector as described in step (1) is pET-32a (+)；

Expression cell as described in step (1) is e. coli bl21 (DE3)；

The culture medium of culture described in step (2) is the LB liquid medium containing 100 μ g/ml ampicillins；

Specific step is as follows for induction described in step (2): adding when the cell containing recombinant vector grows to OD600=1.0 Enter IPTG to final concentration 0.8mM, 20 DEG C, cultivate 20h under 200 r/min.

7. aminopeptidase described in claim 1 is preparing the application in flavor peptide.

8. aminopeptidase according to claim 7 is preparing the application in flavor peptide, it is characterised in that include the following steps: by The aminopeptidase is added in the hydrolysis reaction system containing protein substrate, carries out enzymatic hydrolysis reaction under the conditions of 25~35 DEG C 2~6 h obtain corresponding flavor peptide.