CN112961848B

CN112961848B - Novel aminopeptidase and soluble expression method thereof

Info

Publication number: CN112961848B
Application number: CN202110204785.4A
Authority: CN
Inventors: 刘伟治; 律倩倩; 郑智
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2021-02-24
Filing date: 2021-02-24
Publication date: 2022-09-09
Anticipated expiration: 2041-02-24
Also published as: CN112961848A

Abstract

The invention provides an aminopeptidase O and a soluble expression method thereof, wherein the amino acid sequence of the aminopeptidase O is SEQ OD NO 1; the nucleotide sequence of the coding gene is SEQ ID NO. 2. The invention realizes the soluble expression of the aminopeptidase O in the escherichia coli, thereby promoting the industrial application of the aminopeptidase O. Compared with a eukaryotic expression system, the escherichia coli is a common prokaryotic in-vitro recombinant expression system and has the advantages of short fermentation period, low production and preparation cost and the like; and the soluble expression of active aminopeptidase can effectively avoid activity loss caused by inclusion body renaturation and complex inclusion body renaturation steps, thereby reducing the preparation cost of enzyme.

Description

Novel aminopeptidase and soluble expression method thereof

Technical Field

The invention belongs to the technical field of protease screening and recombinant expression, and particularly relates to an aminopeptidase and a soluble expression method thereof.

Background

Aminopeptidases are a class of exoproteases that selectively cleave amino acid residues from the N-terminus of proteins and polypeptides to produce free amino acids. Aminopeptidases have found wide use in the food and pharmaceutical industries where they are used to debitter food, improve food flavor, and also as tool enzymes for protein sequencing along with other proteases. The classical classification based on the commonly used MEROPS database (https:// www.ebi.ac.uk/MEROPS /) relies on the sensitivity of the active site of aminopeptidases, including the families of serine aminopeptidases, cysteine aminopeptidases and metalloaminopeptidases, to inhibitors. It is well known that the active site of a protein determines the specificity of its catalytic substrate, and therefore there is an urgent need to develop aminopeptidases with different substrate-binding active sites to obtain aminopeptidases with different degradation properties to meet the application requirements.

Among the family of metalloaminopeptidases M1, Aminopeptidase O (AMPO) is a novel class of undecharacterized aminopeptidases. The two families most similar to Aminopeptidase O are respectively Aminopeptidase B (APB) and Leukotriene A4 hydrolase (Leukotriene A-4 hydrosase, LTA4H), with similarities of 23.5% and 20%, respectively. The above aminopeptidases all comprise three domains, the N-terminal domain of M1 (having a function of directing folding), the M1 domain (catalytic domain) and the leukotriene hydrolysis domain. The catalytic domain includes HEXXHX18E and GXMENX conserved modules; among other metalloaminopeptidases, HEXXHX18E was demonstrated on metalloaminopeptidases as a binding domain for zinc ions. Two of the histidines and the following glutamates are believed to interact directly with zinc ions, the former glutamates being the active centers of acid-base catalysis; the conserved sequence considered to be involved in substrate binding and stably associated with the substrate in aminopeptidase O was changed to LGMASP, and other amino acid residues were significantly different except for the conservation of methionine (FIG. 1), suggesting that the influence on the substrate specificity and the binding state of the substrate to the enzyme was exerted.

But until now, aminopeptidase O cannot be expressed in Escherichia coli in a soluble manner, which greatly limits the industrial application thereof.

Disclosure of Invention

The invention aims to provide aminopeptidase O and a soluble expression method thereof, thereby making up the defects of the prior art.

The invention firstly provides an aminopeptidase O, the amino acid sequence of which is as follows:

METQLDPMKDDLPLMANTSYMLVKHYILDLDVDFESKVIEGIIVLFFETGSRYKKTSSTGKGSCQSQFGETCKMRASELCHTPVTNVSACSSKTEYNDFAVCGKGEEDTSDKNGNHSNKEQASGISSSKDCCDIENHGNKDFLLVLDCCDLSVLKVEEVDVAAVSGIEKFTRSAELTDVSKELENLRNQIVHELVTLPADRWKEQLYYFTRCSQAPGCGELLFTTGTWSLEIRKSGIQTPTDFPHAIRIWYKTKPEGRSVTWTTDQSGRPCVYTMGSPINNRALFPCQEPPIALSTWQASVRTAAGFVVLMSGENSAEPVQLREGSLSWYYYVTMPMPASTFTIAVGCWQEVKQQSFTAAIQTNIEFSLPSSQADFRWHEEICGHLEYPCRFQNPAARLQAVIPYRVFAPWCLMEHCEECLLQLIPQCLSAAYTTLGTHPFSRLDVLIVPSNFSSLGMASPHIIFLSQSVLPGGSHLCGTRLCHEIAHAWFGLAIGARDWTEEWISEGFATFLEDIFWARAQQGALLRWRRLRDEVQNSEEELQVLRPKKESTGELSESGASVVKHGLKAEKIFMQVHYLKGYFLLRSLARTIGEASYLASLRKFVHRFHGQLVLSQDFLCMLLEDIPEQKKSELTVESIFQNWLDTSGIPKPLLEEGETWKECQLVRQVSGEVTKWIQTNQRIRKSGKKKRKQDEVVFQKLLPDQLVLLLEYLLEEKTLCPRILQCLEKTYQLREQDAEVRHRWCELVVKHKYVPGYGDVEKFLREDQAMGVYLYGELMVNEDAKQQELAHKCFAAAREHMDASSAKVVAEMLF(SEQ OD NO:1)；

the present invention also provides a gene encoding the aminopeptidase O as described above, which has a nucleotide sequence as follows:

ATGGAAACCCAGCTTGATCCGATGAAAGATGACCTGCCGCTGATGGCGAACACCTCTTATATGCTGGTTAAACACTACATCCTGGACCTGGACGTTGATTTTGAATCTAAAGTGATTGAAGGCATCATTGTTCTGTTTTTCGAAACTGGTTCCCGTTATAAAAAGACCTCTAGCACCGGTAAAGGTAGCTGCCAGAGCCAGTTCGGCGAAACCTGCAAAATGCGTGCGTCTGAACTGTGCCACACCCCGGTGACCAACGTTAGCGCTTGCTCTAGCAAAACCGAATACAACGACTTCGCGGTTTGTGGTAAAGGTGAAGAAGATACCTCCGATAAAAACGGTAACCACTCTAACAAAGAACAGGCGTCTGGCATTAGCAGCAGCAAAGACTGCTGCGATATTGAAAACCACGGTAACAAAGACTTCCTGCTGGTGCTGGATTGCTGCGACCTGAGCGTCCTGAAAGTTGAAGAAGTTGACGTTGCTGCCGTTAGCGGCATTGAAAAATTCACCCGCTCCGCGGAACTGACCGATGTGAGCAAAGAACTGGAAAACCTGCGTAACCAGATCGTGCATGAACTGGTGACTCTGCCGGCGGACCGCTGGAAAGAACAGCTGTACTACTTTACTCGCTGCAGCCAGGCACCGGGCTGCGGCGAACTGCTGTTCACCACCGGCACCTGGTCTCTGGAAATCCGTAAATCTGGCATCCAGACCCCGACCGATTTTCCGCACGCTATTCGTATTTGGTACAAAACCAAACCGGAAGGTCGTAGCGTAACCTGGACCACTGATCAATCCGGCCGTCCGTGCGTCTACACCATGGGTTCCCCGATCAACAACCGTGCGCTGTTCCCGTGCCAGGAACCACCGATCGCACTGAGCACCTGGCAGGCCTCCGTGCGTACCGCAGCAGGCTTTGTTGTTCTGATGAGCGGTGAAAACTCTGCGGAACCGGTGCAGCTGCGTGAAGGTTCCCTTAGCTGGTACTATTACGTTACTATGCCGATGCCAGCTTCCACCTTCACCATCGCCGTGGGTTGTTGGCAGGAAGTTAAACAGCAGTCTTTCACCGCGGCGATCCAGACTAACATCGAATTCTCCCTGCCGAGCTCTCAGGCAGACTTCCGTTGGCACGAAGAAATCTGCGGCCACTTGGAATACCCGTGCCGCTTCCAGAACCCGGCGGCGCGCCTGCAGGCGGTGATCCCGTATCGTGTGTTTGCGCCGTGGTGTCTGATGGAACACTGTGAGGAATGCCTGCTGCAGCTGATTCCGCAATGTCTGAGCGCGGCTTACACCACACTGGGCACCCACCCGTTTAGCCGCCTGGATGTGCTGATCGTTCCGAGCAACTTCAGCTCTCTCGGTATGGCGTCCCCGCATATCATTTTCCTGTCTCAGTCCGTTCTGCCGGGCGGTTCTCACCTGTGCGGCACTCGTCTGTGCCACGAAATCGCACATGCATGGTTCGGCCTGGCTATTGGCGCGCGTGACTGGACCGAAGAATGGATTAGTGAAGGCTTCGCGACCTTTCTGGAAGATATCTTCTGGGCACGTGCTCAGCAGGGCGCACTGCTGCGTTGGCGTCGTCTGCGCGACGAAGTTCAGAACAGCGAAGAAGAACTGCAGGTGCTGCGTCCGAAAAAAGAATCCACCGGTGAACTGTCGGAATCTGGTGCGAGCGTTGTGAAACATGGCCTGAAAGCAGAAAAAATCTTTATGCAGGTTCACTACCTGAAAGGCTACTTCCTGCTGCGCTCTCTGGCGCGCACCATCGGTGAGGCGTCCTACCTGGCTTCCCTGCGTAAATTCGTTCATCGCTTTCACGGCCAGTTGGTGCTGAGCCAGGATTTCCTGTGCATGCTGCTGGAAGATATCCCGGAACAGAAAAAATCCGAACTGACCGTTGAATCTATCTTCCAGAACTGGCTGGATACCAGCGGTATCCCGAAACCGCTGCTGGAAGAAGGCGAAACTTGGAAAGAATGCCAGCTGGTTCGTCAGGTTAGCGGCGAAGTTACCAAATGGATTCAGACCAACCAGCGCATCCGTAAATCTGGCAAAAAGAAACGTAAACAGGATGAAGTAGTTTTCCAGAAACTGCTGCCGGATCAGCTGGTGCTGCTGCTGGAATATCTGCTGGAAGAAAAAACTCTGTGCCCGCGCATCCTGCAGTGTCTGGAAAAAACTTATCAGCTGCGTGAACAGGATGCGGAAGTGCGTCACCGCTGGTGTGAACTGGTTGTTAAACATAAATATGTTCCTGGCTACGGTGATGTTGAAAAATTCCTGCGTGAAGATCAGGCTATGGGTGTTTACCTGTACGGTGAACTGATGGTTAACGAAGATGCGAAACAGCAGGAACTGGCACATAAATGCTTCGCAGCAGCGCGTGAACACATGGACGCAAGCAGCGCAAAAGTTGTGGCGGAAATGCTGTTC(SEQ ID NO:2)；

the invention also provides a recombinant expression vector, which is constructed by inserting the nucleotide fragment for coding the aminopeptidase O into the vector;

the vector is a prokaryotic expression vector pMCSG9 vector;

the invention also provides a method for expressing the aminopeptidase O, which is to ferment after transferring the recombinant expression vector into host bacteria and to express the aminopeptidase O in a recombinant manner;

the host bacterium is escherichia coli.

The invention also provides the application of the aminopeptidase O, which is used for degrading polypeptide to generate glutamic acid Glu; glu is the most umami amino acid in common flavor amino acids, so the aminopeptidase with the degradation of Glu has unique application potential in flavor food processing.

The invention realizes the soluble expression of the aminopeptidase O in the escherichia coli, thereby promoting the industrial application of the aminopeptidase O. Compared with a eukaryotic expression system, the escherichia coli is a common prokaryotic in-vitro recombinant expression system and has the advantages of short fermentation period, low production and preparation cost and the like; and the soluble expression of active aminopeptidase can effectively avoid activity loss caused by inclusion body renaturation and complex inclusion body renaturation steps, thereby reducing the preparation cost of enzyme.

Drawings

FIG. 1: comparison of partial peptidase of M1 peptidase family with related sequence of zinc ion action (paAMPO fragment 403-550), wherein the first four protein source organisms are human, the pre-name identification code is the identification code of the enzyme in uniprot database, and the post-name is the corresponding name (RNPL 1: Aminopeptidase RNPEPL1, Aminopeptidase B-like peptidase); XP-009326408.1 is the NCBI Adeli penguin aminopeptidase O sequence identifier code. Three black bold line markers before and after the GXMENX and HEXXHX18E conserved motifs and comparison NCBI aminopeptidase key modules, respectively, black markers are key amino acids that have been shown to be associated with substrate binding and activity;

FIG. 2: a diagram for optimizing preamplification and induction conditions of paAMPO, wherein (a) a pET28a vector expresses paAMPO, M is Marker, 1 is before induction, 2 is after induction, 3 is soluble protein, and 4 is inclusion body protein; (b) the pMCSG9 vector optimizes expression paAMPO, M is Marker, and the induction condition is that OD600 is 0.4IPTG 0.4 mM; 1-1 is inclusion body, 1-2 is soluble protein.

FIG. 3: MBP-paAMPO fusion protein expression and enzymology detection maps, wherein a map a is pMCSG9-paAMPO crude enzyme, and the predicted size of MBP-paAMPO is 140 kDa; m is Marker, lane 1 is soluble protein, lane 2 is inclusion body, lane 3 is crude enzyme; panel b shows the activities of paAMPO crude enzyme on Arg-pNA, Leu-pNA and Glu-pNA, as a control, unloaded crude enzyme, relative activity ═ crude enzyme activity/unloaded crude enzyme activity.

FIG. 4: q column elution and Glu-pNA activity profiles, wherein (a) SDS-PAGE of the Q column linear elution protein, M is Marker, lane 1 crude enzyme, i.e., before loading, and lane 2 combined and eluted, lane 3 is 200mM NaCl elution protein, and lanes 4-10 are 200mM NaCl linear elution protein, respectively designated as Q1-Q7. The strip is a cutting adhesive tape for mass spectrum detection; (b)200-500mM linear elution sample (Q1-Q7) for Glu-pNA activity changes.

FIG. 5 is a schematic view of: the coverage map of the characteristic peptide is detected by the protein shotgun mass spectrum, the whole sequence is the complete sequence of paAMPO, and the detection fragment is in italic background.

Detailed Description

The present invention will be described in detail below with reference to examples and the accompanying drawings.

Example 1

A new aminopeptidase is obtained after analysis and splicing prediction of the genome data of the Adeli penguin, and is named as AMPO _ Pygoscelis adelaiae (paAMPO), and the amino acid sequence of the aminopeptidase is SEQ ID NO. 1. The amino acid sequence of the aminopeptidase paAMPOD, which lacks 12 amino acid residues in the catalytic domain M1 domain (fig. 1) compared to the protein sequence on NCBI (XP — 009326408.1), is presumed to have an important effect on the expression and activity of the enzyme.

The optimized nucleotide sequence of the paAMPO coding gene is compared with the nucleotide sequence Genbank on NCBI: XM _009328133) had 73.94% sequence homology.

Example 2: screening and optimization of different expression vectors

In view of the difficulty of aminopeptidase expression and the need for industrial application, various expression tags, His-tag and MBP-tag, and expression conditions were selected

Synthesizing genes: synthesizing the gene of paAMPO according to the optimized nucleic acid sequence information, and designing to add a BamHI enzyme digestion sequence at the front end of the synthesized gene and add an XhoI enzyme digestion sequence after a stop codon at the rear end by analyzing sequence enzyme digestion site information and alternative carrier information.

Construction of an expression strain: PaAMPO with a restriction enzyme sequence was digested with BamH I/Xho I, Gel was cut after agarose electrophoresis, recovered using Gel Extraction Kit (OMEGA), cloned into pET28a and pMCSG9 using T4 ligase (Takara) to construct pET28a-paAMPO and pMCSG9-paAMPO, under conditions of 16 ℃ for 4 hours, and E.coil BL21(DE3) was transformed. Then, solid LB medium screening with kanamycin (Kan) and ampicillin (Amp), respectively, was performed, single clones were selected, colony PCR was verified, the amplification enzyme was detected using 2 XPCR mix (Dongsheng organism), and the primers were universal primers for pET28a and pMCSG9 (T7& T7ter, Bio/S), as shown in Table 1 below. After culturing correctly screened strains, the strains are stored in 20% glycerol and stored at-80 ℃.

Table 1: reaction System Table of PCR

2×PCR mix	5μl
		Primer T7 (10. mu.M)	1μl
Primer and method for producing the sameT7 ter(10μM)	1μl
		Double distilled water	3μl

Pre-expression and expression optimization: inoculating the constructed expression strain into a liquid culture medium (pET28a corresponding to Kan, pMCSG9 corresponding to Amp) containing corresponding antibiotics, carrying out shaking culture at 37 ℃ until the OD600 of a bacterial liquid is 0.6-0.8, adding IPTG (isopropyl-beta-D-thiogalactoside, galactose analogue) with the final concentration of 0.4mM for induction, reducing the culture temperature to 20 ℃, and carrying out overnight culture. Centrifuging at 3000 Xg for 30min to collect thallus, adding lysis buffer (20mM Tris-HCl pH 8.0), suspending, ultrasonic crushing, centrifuging at 12000 Xg for 30min to obtain supernatant as soluble protein, and precipitating as inclusion body protein. The results of the assay suggested that pET28a-paAMPO was expressed in inclusion bodies (FIG. 2 a). Whereas pMCSG9-paAMPO had significant soluble expression (FIG. 2 b).

The conditions for expression of pMCSG9-paAMPO are as follows: culturing at 37 deg.C until OD600 is 0.6-0.8, adding IPTG to a final concentration of 0.4mM, inducing, transferring to 20 deg.C, and culturing overnight.

Example 3: activity detection of paAMPO protein

To examine the activity of the recombinant protein, according to a general method for assaying aminopeptidase activity, aa-pNA (amino acid p-nitroaniline) was used as a substrate, and the acidic amino acid p-nitroaniline glutamate (Glu-pNA), the neutral amino acid p-nitroaniline leucine (Leu-pNA), and the basic amino acid p-nitroaniline arginine (Arg-pNA) were used as substrates for assaying the activity and determining the specificity of the substrate, depending on the acidity or basicity of the amino acid.

1. Expressing the recombinant protein according to the optimized conditions, centrifuging at 3000 Xg for 30min to collect thallus, adding lysis buffer (20mM Tris-HCl pH 8.0), placing the thallus suspension on an ultrasonic pulverizer to perform thallus ultrasonic wave (New glossy ganoderma JY 92-IIN) crushing at 2s and 4s according to a program of 50% power for 15min, centrifuging at 12000 Xg for 30min, collecting supernatant soluble protein, dialyzing in the lysis buffer for 2h, and centrifuging at 12000 Xg for 10min to obtain a protein liquid which is regarded as crude enzyme shown in figure 3 (a).

2. Substrate dissolution: Glu-pNA was dissolved in 20mM Tris-HCl pH8.0 to a final concentration of 2.5 mM; Arg-pNA and Leu-pNA were dissolved in water to a final concentration of 10.0 mM.

3. And (3) activity detection: the activity was measured according to the following 200. mu.l reaction: 50mM Tris-HCl pH8.0, 200mM NaCl, 0.25mM aa-pNA, 20. mu.l crude enzyme. The reaction was carried out at 37 ℃ for 30min, and the absorbance at 405nm was measured. Crude enzyme prepared from pMCSG9 in which soluble protein was expressed in an empty state (hereinafter, referred to as empty crude enzyme) in the same manner and in the same amount as the cells (see FIG. 3 (b)), paAMPO showed the most different activity against Glu-pNA from the control, which also suggests that the crude enzyme is different from the empty crude enzyme, indicating that paAMPO has Glu-pNA activity, which is an aminopeptidase having Glu substrate specificity.

4. Ion exchange chromatography purification and mass spectrometry identification:

obtaining a crude enzyme with specificity to Glu-pNA, and needing to improve the purity; soluble expression of paAMPO was further determined by mass spectrometry.

And (3) chromatographic column determination: both paAMPO and MBP-paAMPO are predicted by the website (https:// web. expasy. org/protparam /) to have pIs less than 7 in a buffer at pH8, so a strong anion exchange chromatography column is used: HiTrap Q XL (1ml, GE).

5. HiTrap Q XL chromatography: the chromatographic procedure was completed on an AKTA Rapid protein purification apparatus (Pure 25, GE). The column was first equilibrated with 10 Column Volumes (CV) in combination with buffer; after the crude enzyme was prepared as above, it was filtered through a 0.22 μm filter membrane and then loaded at 1 ml/min; washing the column with binding buffer until a baseline level of UV280 to remove proteins unbound or loosely bound to the column; the column was then eluted with elution buffer 1(20mM Tris-HCl pH8.0, 200mM NaCl) and the protein eluate (200 crude enzyme) was collected; after the baseline level, the column was eluted linearly for 5CV with 200mM-500mM NaCl, and the protein was collected in 0.5ml aliquots per tube (Q1-Q7). The samples that retained the critical step were electrophoresed and the results are shown in FIG. 4 (a). Activity determination the activity was concentrated in 200mM-500mM linear elution samples and the results are shown in FIG. 4 (b). But the predicted size fraction (140kDa) was not contained in this fraction, which was presumed to be mostly a misfolded fusion protein. Based on the apparent increase in size of MBP (42kDa) molecules, it is speculated that MBP-paAMPO is degraded to produce a MBP-free paAMPO protein of about 100kDa (FIG. 4(a) band).

Protein shotgun mass spectrometric detection: to identify whether the "band" component is a paAMPO component, the "band" was subjected to a protein shotgun mass spectrometric detection. Cutting a Coomassie dyeing adhesive tape after polyacrylamide electrophoresis, detecting a substance spectrum after trypsin enzymolysis, identifying and analyzing by using a built-in software, namely a protocol discover 2.4(Thermo Scientific) library of Sequest HT, searching an entire sequence with a target of paAMPO, and marking characteristic peptides, wherein the lost enzyme cutting site is less than or equal to 2. The mass spectrum result shows that 37 characteristic peptides are detected in the band, the coverage rate of the characteristic peptides on the paAMPO is 41 percent (as shown in figure 5), and the expression of the paAMPO at the position of the band can be proved.

In conclusion, the invention realizes the soluble expression of aminopeptidase O in Escherichia coli for the first time, and determines the activity of the aminopeptidase O on Glu-pNA, which is different from aminopeptidase B and leukotriene A4 hydrolase with the highest sequence similarity.

Sequence listing

<110> China oceanic university

<120> a novel aminopeptidase and soluble expression method thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 815

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Glu Thr Gln Leu Asp Pro Met Lys Asp Asp Leu Pro Leu Met Ala

1 5 10 15

Asn Thr Ser Tyr Met Leu Val Lys His Tyr Ile Leu Asp Leu Asp Val

20 25 30

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

35 40 45

Thr Gly Ser Arg Tyr Lys Lys Thr Ser Ser Thr Gly Lys Gly Ser Cys

50 55 60

Gln Ser Gln Phe Gly Glu Thr Cys Lys Met Arg Ala Ser Glu Leu Cys

65 70 75 80

His Thr Pro Val Thr Asn Val Ser Ala Cys Ser Ser Lys Thr Glu Tyr

85 90 95

Asn Asp Phe Ala Val Cys Gly Lys Gly Glu Glu Asp Thr Ser Asp Lys

100 105 110

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

115 120 125

Lys Asp Cys Cys Asp Ile Glu Asn His Gly Asn Lys Asp Phe Leu Leu

130 135 140

Val Leu Asp Cys Cys Asp Leu Ser Val Leu Lys Val Glu Glu Val Asp

145 150 155 160

Val Ala Ala Val Ser Gly Ile Glu Lys Phe Thr Arg Ser Ala Glu Leu

165 170 175

Thr Asp Val Ser Lys Glu Leu Glu Asn Leu Arg Asn Gln Ile Val His

180 185 190

Glu Leu Val Thr Leu Pro Ala Asp Arg Trp Lys Glu Gln Leu Tyr Tyr

195 200 205

Phe Thr Arg Cys Ser Gln Ala Pro Gly Cys Gly Glu Leu Leu Phe Thr

210 215 220

Thr Gly Thr Trp Ser Leu Glu Ile Arg Lys Ser Gly Ile Gln Thr Pro

225 230 235 240

Thr Asp Phe Pro His Ala Ile Arg Ile Trp Tyr Lys Thr Lys Pro Glu

245 250 255

Gly Arg Ser Val Thr Trp Thr Thr Asp Gln Ser Gly Arg Pro Cys Val

260 265 270

Tyr Thr Met Gly Ser Pro Ile Asn Asn Arg Ala Leu Phe Pro Cys Gln

275 280 285

Glu Pro Pro Ile Ala Leu Ser Thr Trp Gln Ala Ser Val Arg Thr Ala

290 295 300

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

305 310 315 320

Gln Leu Arg Glu Gly Ser Leu Ser Trp Tyr Tyr Tyr Val Thr Met Pro

325 330 335

Met Pro Ala Ser Thr Phe Thr Ile Ala Val Gly Cys Trp Gln Glu Val

340 345 350

Lys Gln Gln Ser Phe Thr Ala Ala Ile Gln Thr Asn Ile Glu Phe Ser

355 360 365

Leu Pro Ser Ser Gln Ala Asp Phe Arg Trp His Glu Glu Ile Cys Gly

370 375 380

His Leu Glu Tyr Pro Cys Arg Phe Gln Asn Pro Ala Ala Arg Leu Gln

385 390 395 400

Ala Val Ile Pro Tyr Arg Val Phe Ala Pro Trp Cys Leu Met Glu His

405 410 415

Cys Glu Glu Cys Leu Leu Gln Leu Ile Pro Gln Cys Leu Ser Ala Ala

420 425 430

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

435 440 445

Val Pro Ser Asn Phe Ser Ser Leu Gly Met Ala Ser Pro His Ile Ile

450 455 460

Phe Leu Ser Gln Ser Val Leu Pro Gly Gly Ser His Leu Cys Gly Thr

465 470 475 480

Arg Leu Cys His Glu Ile Ala His Ala Trp Phe Gly Leu Ala Ile Gly

485 490 495

Ala Arg Asp Trp Thr Glu Glu Trp Ile Ser Glu Gly Phe Ala Thr Phe

500 505 510

Leu Glu Asp Ile Phe Trp Ala Arg Ala Gln Gln Gly Ala Leu Leu Arg

515 520 525

Trp Arg Arg Leu Arg Asp Glu Val Gln Asn Ser Glu Glu Glu Leu Gln

530 535 540

Val Leu Arg Pro Lys Lys Glu Ser Thr Gly Glu Leu Ser Glu Ser Gly

545 550 555 560

Ala Ser Val Val Lys His Gly Leu Lys Ala Glu Lys Ile Phe Met Gln

565 570 575

Val His Tyr Leu Lys Gly Tyr Phe Leu Leu Arg Ser Leu Ala Arg Thr

580 585 590

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

595 600 605

Phe His Gly Gln Leu Val Leu Ser Gln Asp Phe Leu Cys Met Leu Leu

610 615 620

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

625 630 635 640

Phe Gln Asn Trp Leu Asp Thr Ser Gly Ile Pro Lys Pro Leu Leu Glu

645 650 655

Glu Gly Glu Thr Trp Lys Glu Cys Gln Leu Val Arg Gln Val Ser Gly

660 665 670

Glu Val Thr Lys Trp Ile Gln Thr Asn Gln Arg Ile Arg Lys Ser Gly

675 680 685

Lys Lys Lys Arg Lys Gln Asp Glu Val Val Phe Gln Lys Leu Leu Pro

690 695 700

Asp Gln Leu Val Leu Leu Leu Glu Tyr Leu Leu Glu Glu Lys Thr Leu

705 710 715 720

Cys Pro Arg Ile Leu Gln Cys Leu Glu Lys Thr Tyr Gln Leu Arg Glu

725 730 735

Gln Asp Ala Glu Val Arg His Arg Trp Cys Glu Leu Val Val Lys His

740 745 750

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

755 760 765

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

770 775 780

Ala Lys Gln Gln Glu Leu Ala His Lys Cys Phe Ala Ala Ala Arg Glu

785 790 795 800

His Met Asp Ala Ser Ser Ala Lys Val Val Ala Glu Met Leu Phe

805 810 815

<210> 2

<211> 2445

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<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggaaaccc agcttgatcc gatgaaagat gacctgccgc tgatggcgaa cacctcttat 60

atgctggtta aacactacat cctggacctg gacgttgatt ttgaatctaa agtgattgaa 120

ggcatcattg ttctgttttt cgaaactggt tcccgttata aaaagacctc tagcaccggt 180

aaaggtagct gccagagcca gttcggcgaa acctgcaaaa tgcgtgcgtc tgaactgtgc 240

cacaccccgg tgaccaacgt tagcgcttgc tctagcaaaa ccgaatacaa cgacttcgcg 300

gtttgtggta aaggtgaaga agatacctcc gataaaaacg gtaaccactc taacaaagaa 360

caggcgtctg gcattagcag cagcaaagac tgctgcgata ttgaaaacca cggtaacaaa 420

gacttcctgc tggtgctgga ttgctgcgac ctgagcgtcc tgaaagttga agaagttgac 480

gttgctgccg ttagcggcat tgaaaaattc acccgctccg cggaactgac cgatgtgagc 540

aaagaactgg aaaacctgcg taaccagatc gtgcatgaac tggtgactct gccggcggac 600

cgctggaaag aacagctgta ctactttact cgctgcagcc aggcaccggg ctgcggcgaa 660

ctgctgttca ccaccggcac ctggtctctg gaaatccgta aatctggcat ccagaccccg 720

accgattttc cgcacgctat tcgtatttgg tacaaaacca aaccggaagg tcgtagcgta 780

acctggacca ctgatcaatc cggccgtccg tgcgtctaca ccatgggttc cccgatcaac 840

aaccgtgcgc tgttcccgtg ccaggaacca ccgatcgcac tgagcacctg gcaggcctcc 900

gtgcgtaccg cagcaggctt tgttgttctg atgagcggtg aaaactctgc ggaaccggtg 960

cagctgcgtg aaggttccct tagctggtac tattacgtta ctatgccgat gccagcttcc 1020

accttcacca tcgccgtggg ttgttggcag gaagttaaac agcagtcttt caccgcggcg 1080

atccagacta acatcgaatt ctccctgccg agctctcagg cagacttccg ttggcacgaa 1140

gaaatctgcg gccacttgga atacccgtgc cgcttccaga acccggcggc gcgcctgcag 1200

gcggtgatcc cgtatcgtgt gtttgcgccg tggtgtctga tggaacactg tgaggaatgc 1260

ctgctgcagc tgattccgca atgtctgagc gcggcttaca ccacactggg cacccacccg 1320

tttagccgcc tggatgtgct gatcgttccg agcaacttca gctctctcgg tatggcgtcc 1380

ccgcatatca ttttcctgtc tcagtccgtt ctgccgggcg gttctcacct gtgcggcact 1440

cgtctgtgcc acgaaatcgc acatgcatgg ttcggcctgg ctattggcgc gcgtgactgg 1500

accgaagaat ggattagtga aggcttcgcg acctttctgg aagatatctt ctgggcacgt 1560

gctcagcagg gcgcactgct gcgttggcgt cgtctgcgcg acgaagttca gaacagcgaa 1620

gaagaactgc aggtgctgcg tccgaaaaaa gaatccaccg gtgaactgtc ggaatctggt 1680

gcgagcgttg tgaaacatgg cctgaaagca gaaaaaatct ttatgcaggt tcactacctg 1740

aaaggctact tcctgctgcg ctctctggcg cgcaccatcg gtgaggcgtc ctacctggct 1800

tccctgcgta aattcgttca tcgctttcac ggccagttgg tgctgagcca ggatttcctg 1860

tgcatgctgc tggaagatat cccggaacag aaaaaatccg aactgaccgt tgaatctatc 1920

ttccagaact ggctggatac cagcggtatc ccgaaaccgc tgctggaaga aggcgaaact 1980

tggaaagaat gccagctggt tcgtcaggtt agcggcgaag ttaccaaatg gattcagacc 2040

aaccagcgca tccgtaaatc tggcaaaaag aaacgtaaac aggatgaagt agttttccag 2100

aaactgctgc cggatcagct ggtgctgctg ctggaatatc tgctggaaga aaaaactctg 2160

tgcccgcgca tcctgcagtg tctggaaaaa acttatcagc tgcgtgaaca ggatgcggaa 2220

gtgcgtcacc gctggtgtga actggttgtt aaacataaat atgttcctgg ctacggtgat 2280

gttgaaaaat tcctgcgtga agatcaggct atgggtgttt acctgtacgg tgaactgatg 2340

gttaacgaag atgcgaaaca gcaggaactg gcacataaat gcttcgcagc agcgcgtgaa 2400

cacatggacg caagcagcgc aaaagttgtg gcggaaatgc tgttc 2445

Claims

1. An aminopeptidase O, wherein the amino acid sequence of the aminopeptidase O is SEQ ID NO. 1.

2. A nucleic acid fragment encoding the aminopeptidase O of claim 1.

3. The nucleic acid fragment of claim 2, wherein the sequence of said nucleic acid fragment is SEQ ID No. 2.

4. A recombinant expression vector, wherein the recombinant expression vector is an expression vector carrying the nucleic acid fragment of claim 2.

5. The recombinant expression vector according to claim 4, wherein the expression vector is prokaryotic expression vector pMCSG9 vector.

6. A method for expressing the aminopeptidase O of claim 1, which comprises transferring the recombinant expression vector of claim 4 into a host bacterium, and performing recombinant expression to produce the aminopeptidase O.

7. The method of claim 6, wherein the host bacterium is Escherichia coli.

8. Use of the aminopeptidase O according to claim 1 for degrading a polypeptide to produce Glu glutamate.