CN106755220A - The method for preparing nisin - Google Patents
The method for preparing nisin Download PDFInfo
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- CN106755220A CN106755220A CN201510819391.4A CN201510819391A CN106755220A CN 106755220 A CN106755220 A CN 106755220A CN 201510819391 A CN201510819391 A CN 201510819391A CN 106755220 A CN106755220 A CN 106755220A
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Abstract
Method the invention discloses nisin is prepared, the method is comprised the following steps：Using the albumen of microbial expression first, to obtain nisin precursor peptide；The nisin precursor peptide is modified using NisB albumen and NisC albumen, to obtain the nisin precursor peptide by modifying；The nisin precursor peptide by modification is digested in vitro using the albumen for being capable of identify that NisP cleavage sites, to obtain nisin.There is no bioactivity using the resulting albumen in microbial body of the method, the rupture of cell will not be caused, and, the method of the present invention can break the cellular stress of the fully synthetic Nisin techniques of existing microorganism, not only yield is high for the product for obtaining, purity is also purer, is suitable to industrialization large-scale production.
Description
Technical field
Method the present invention relates to prepare nisin.
Background technology
It is that Buddhist nun is pungent that nisin (Nisin) is also known as nisin or transliteration, is a kind of polypeptide that streptococcus lactis is produced
Material, is made up of 34 amino acid residues.Because nisin can suppress most of gram-positive bacteriums, and to bud
The spore of spore bacillus has strong inhibitory action, therefore is widely used in food service industry as food preservative.In people after edible
Amino acid is hydrolyzed into quickly under the physiological pH condition and α of body-chymotrypsin protein enzyme effect, will not change normal in human body intestinal canal
Flora and the generation resistance problem that such as other antibiotic occur, more cross tolerance will not occur with other antibiotic, be a kind of
Efficiently, it is nontoxic, safely, the antiseptics for natural food that has no side effect.
The production of current Nisin products is fermented by lactic acid bacteria, can be destroyed yet with ripe Nisin
The cell membrane of bacterium, causes cell rupture, so as to cause the process yields currently with the fully synthetic Nisin of microorganism relatively low.And
And, although can protect bacterium in itself in the presence of its immunologic mechanism, i.e. NisEFG and NisI albumen in Nisin production bacterial spawns,
But this immunologic mechanism is limited, it is impossible to change the relatively low present situation of the fully synthetic Nisin process yields of microorganism.
Thus, current process for preparing lactostreptostacin still has much room for improvement.
The content of the invention
It is contemplated that at least solving one of technical problem present in prior art.Therefore, it is an object of the present invention to carry
Go out a kind of suitable large-scale culture, original cellular stress can be broken, obtain product yield and purity Nisin producers high
Formula.
The present invention is to be based on the following discovery of inventor and complete：
The fully synthetic Nisin techniques of current microorganism, the route of synthesis in microbial body is as follows：It is (or other by nisA/Z genes
Gene, such as nisQ, nisF, nisU) encoding precursor peptide (Nisin precursor), the precursor peptide is again by nisB and nisC
NisB the and NisC albumen of gene code is modified precursor peptide, and wherein NisB is act as to specific serine and Soviet Union's ammonia
Acid is dehydrated, and NisC act as forming wool sulphur ring, then by nisP gene outcome NisP albumen to the albumen after modification
Cut, eventually formed the NisinA/NisinZ products of maturation.
Inventor's discovery, the semi-matured Nisin precursor peptides of great expression in microbial body, then by these immature precursors
Peptide in vitro using nisP albumen or other can recognize that the albumen of nisP cleavage sites is digested, you can largely had work
Property ripe Nisin, and albumen (i.e. semi-matured Nisin precursor peptides) resulting in microbial body is without bioactivity,
The rupture of cell will not be caused, the bottleneck by the fully synthetic Nisin of microorganism can be broken through.This mode of production is more suitable for big
Scale evaluation, can break original cellular stress, and not only yield is high for the product for obtaining, purity is also purer, be a kind of brand-new
The Nisin modes of production.
Thus, according to an aspect of the present invention, the invention provides a kind of method for preparing nisin.According to this hair
Bright embodiment, the method is comprised the following steps：Nisin precursor peptide is obtained using microbial expression；Using NisB
Albumen and NisC albumen are modified the nisin precursor peptide, to obtain the nisin by modifying
Precursor peptide；And the nisin precursor peptide by modification is entered using the albumen for being capable of identify that NisP cleavage sites
The external enzymolysis of row, to obtain nisin, wherein, the NisB albumen has SEQ ID NO：Ammonia shown in 1
Base acid sequence, the NisC albumen has SEQ ID NO：Amino acid sequence shown in 2.
It is surprisingly found by the inventors that, using the method for preparing nisin of the invention, great expression half in microbial body
Ripe Nisin precursor peptides, are then carried out using the albumen for being capable of identify that NisP cleavage sites to semi-matured Nisin precursor peptides
External enzymolysis, you can obtain a large amount of active ripe Nisin, and albumen resulting in microbial body (i.e. half ripe
Nisin precursor peptides) without bioactivity, the rupture of cell will not be caused, can break through by the fully synthetic Nisin of microorganism
Bottleneck.Also, embodiments in accordance with the present invention, the method for the present invention can break the fully synthetic Nisin techniques of existing microorganism
Cellular stress, not only yield is high for the product for obtaining, purity is also purer, is a kind of brand-new Nisin modes of production, and fit
Mass produced in industrialization.
Wherein, the sequence of NisB albumen is as follows：
MIKSSFKAQPFLVRNTILSPNDKRSFTEYTQVIETVSKNKVFLEQLLLANPKLYDVMQ
KYNAGLLKKKRVKKLFESIYKYYKRSYLRSTPFGLFSETSIGVFSKSSQYKLMGKTTKGIR
LDTQWLIRLVHKMEVDFSKKLSFTRNNANYKFGDRVFQVYTINSSELEEVNIKYTNVYQII
SEFCENDYQKYEDICETVTLCYGDEYRELSEQYLGSLIVNHYLISNLQKDLLSDFSWNTFL
TKVEAIDEDKKYIIPLKKVQKFIQEYSEIEIGEGIEKLKEIYQEMSQILENDNYIQIDLISDSEI
NFDVKQKQQLEHLAEFIGNTTKSVRRTYLDDYKDKFIEKYGVDQEVQITELFDSTFGIGAP
YNYNHPRNDFYESEPSTLYYSEEEREKYLSMYVEAVKNHNVINLDDLESHYQKMDLEKK
SELQGLELFLNLAKEYEKDIFILGDIVGNNNLGGASGRFSALSPELTSYHRTIVDSVERENE
NKEITSCEIVFLPENIRHANVMHTSIMRRKVLPFFTSTSHNEVLLTNIYIGIDEKEKFYARDIS
TQEVLKFYITSMYNKTLFSNELRFLYEISLDDKFGNLPWELIYRDFDYIPRLVFDEIVISPAK
WKIWGRDVNSKITIRELIQSKEIPKEFYIVNGDNKVYLSQENPLDMEILESAIKKSSKRKDFI
ELQEYFEDENIINKGQKGRVADVVVPFIRTRALGNEGRAFIREKRVSVERREKLPFNEWLY
LKLYISINRQNEFLLSYLPDIQKIVANLGGNLFFLRYTDPKPHIRLRIKCSDLFLAYGSILEILK
RSRKNRIMSTFDISIYDQEVERYGGFDTLELSEAIFCADSKIIPNLLTLIKDTNNDWKVDDVS
ILVNYLYLKCFFQNDNKKILNFLNLVSPKKVKENVNEKIEHYLKLLKVNNLGDQIFYDKNF
KELKHAIKNLFLKMIAQDFELQKVYSIIDSIIHVHNNRLIGIERDKEKLIYYTLQRLFVSEEY
MK(SEQ ID NO：1),
The sequence of NisC albumen is as follows：
MNKKNIKRNVEKIIAQWDERTRKNKENFDFGELTLSTGLPGIILMLAELKNKDNSKIY
QKKIDNYIEYIVSKLSTYGLLTGSLYSGAAGIALSILHLREDDEKYKNLLDSLNRYIEYFVR
EKIEGFNLENITPPDYDVIEGLSGILSYLLLINDEQYDDLKILIINFLSNLTKENKGLISLYIKS
ENQMSQSESEMYPLGCLNMGLAHGLAGVGCILAYAHIKGYSNEASLSALQKIIFIYEKFEL
ERKKQFLWKDGLVADELKKEKVIREASFIRDAWCYGGPGISLLYLYGGLALDNDYFVDKA
EKILESAMQRKLGIDSYMICHGYSGLIEICSLFKRLLNTKKFDSYMEEFNVNSEQILEEYGD
ESGTGFLEGISGCILVLSKFEYSINFTYWRQALLLFDDFLKGGKRK(SEQ ID NO：2).
In addition, the method for preparing nisin according to the above embodiment of the present invention can also have following additional technology special
Levy：
Embodiments in accordance with the present invention, the nisin precursor peptide is selected from NisA albumen, NisZ albumen, NisQ
At least one of albumen, NisF albumen, NisU albumen and NisU2 albumen, wherein, the NisA albumen has SEQ ID
NO：Amino acid sequence shown in 3, the NisZ albumen has SEQ ID NO：Amino acid sequence shown in 4, the NisQ
Albumen has SEQ ID NO：Amino acid sequence shown in 5, the NisF albumen has SEQ ID NO：Ammonia shown in 6
Base acid sequence, the NisU albumen has SEQ ID NO：Amino acid sequence shown in 7, the NisU2 albumen has SEQ
ID NO：Amino acid sequence shown in 8.Thus, microbial expression NisA albumen, NisZ albumen, NisQ albumen, NisF are made
At least one of albumen, NisU albumen and NisU2 albumen, can effectively obtain nisin precursor peptide.
Specifically, the sequence of NisA albumen is as follows：
MSTKDFNLDLVSVSKKDSGASPRITSISLCTPGCKTGALMGCNMKTATCNCSIHVSK
(SEQ ID NO：3),
The sequence of NisZ albumen is as follows：
MSTKDFNLDLVSVSKKDSGASPRITSISLCTPGCKTGALMGCNMKTATCNCSIHVSK
(SEQ ID NO：4),
The sequence of NisQ albumen is as follows：
MSTKDFNLDLVSVSKTDSGASTRITSISLCTPGCKTGVLMGCNLKTATCNCSVHVSK
(SEQ ID NO：5),
The sequence of NisF albumen is as follows：
MSTKDFNLDLVSVSKKDSGASPRITSISLCTPGCKTGALMGCNMKTATCNCSVHVSK
(SEQ ID NO：6),
The sequence of NisU albumen is as follows：
MSTKDFNLDLVSVSKKDSGASPRITSKSLCTPGCKTGILTGCPLKTATCGCHFG(SEQ
ID NO：7),
The sequence of NisU2 albumen is as follows：
MSTKDFNLDLVSVSKKDSGASPRVTSKSLCTPGCKTGILTGCPLKTATCGCHFG(SEQ
ID NO：8).
Embodiments in accordance with the present invention, the NisA albumen is by SEQ ID NO：Nucleic acid sequence encoding shown in 12, it is described
NisZ albumen is by SEQ ID NO：Nucleic acid sequence encoding shown in 13, the NisQ albumen is by SEQ ID NO：Shown in 14
Nucleic acid sequence encoding, the NisF albumen is by SEQ ID NO：Nucleic acid sequence encoding shown in 15, the NisU albumen
By SEQ ID NO：Nucleic acid sequence encoding shown in 16, the NisU2 albumen is by SEQ ID NO：Nucleic acid sequence shown in 17
Row coding.Thus, protein expression efficiency is high such that it is able to efficiently obtain various nisin precursor peptides.
Specifically, the encoding gene of NisA albumen is nisA genes, and its sequence is as follows：
atgagtacaaaagattttaacttggatttggtatctgtttcgaagaaagattcaggtgcatcaccacgcattacaagtatttcgctatgtaca
cccggttgtaaaacaggagctctgatgggttgtaacatgaaaacagcaacttgtaattgtagtattcacgtaagcaaataa(SEQ ID NO：
12),
The encoding gene of NisZ albumen is nisZ genes, and its sequence is as follows：
ATGAGTACAAAAGATTTTAACTTGGATTTGGTATCTGTTTCGAAGAAAGATTCAGG
TGCATCACCACGCATTACAAGTATTTCGCTATGTACACCCGGTTGTAAAACAGGAGCTC
TGATGGGTTGTAACATGAAAACAGCAACTTGTAATTGTAGTATTCACGTAAGCAAATAA
(SEQ ID NO：13),
The encoding gene of NisQ albumen is nisQ genes, and its sequence is as follows：
ATGAGCACCAAGGACTTCAACCTGGATCTGGTGAGCGTTAGCAAAACCGACAGCG
GCGCGAGCACCCGTATCACCAGCATTAGCCTGTGCACCCCGGGTTGCAAGACCGGCGT
GCTGATGGGTTGCAACCTGAAAACCGCGACCTGCAACTGCAGCGTGCACGTTAGCAA
GTAA(SEQ ID NO：14),
The encoding gene of NisF albumen is nisF genes, and its sequence is as follows：
ATGAGCACCAAGGACTTCAACCTGGATCTGGTGAGCGTTAGCAAGAAAGACAGC
GGTGCGAGCCCGCGTATCACCAGCATTAGCCTGTGCACCCCGGGTTGCAAGACCGGCG
CGCTGATGGGTTGCAACATGAAAACCGCGACCTGCAACTGCAGCGTGCACGTTAGCAA
ATAA(SEQ ID NO：15),
The encoding gene of NisU albumen is nisU genes, and its sequence is as follows：
ATGAGCACCAAGGACTTCAACCTGGATCTGGTGAGCGTTAGCAAGAAAGACAGC
GGTGCGAGCCCGCGTATCACCAGCAAGAGCCTGTGCACCCCGGGTTGCAAAACCGGC
ATTCTGACCGGTTGCCCGCTGAAAACCGCGACCTGCGGTTGCCACTTTGGTTAA(SEQ
ID NO：16),
The encoding gene of NisU2 albumen is nisU2 genes, and its sequence is as follows：
ATGAGCACCAAGGACTTCAACCTGGATCTGGTGAGCGTTAGCAAGAAAGACAGC
GGTGCGAGCCCGCGTGTGACCAGCAAGAGCCTGTGCACCCCGGGTTGCAAAACCGGC
ATCCTGACCGGTTGCCCGCTGAAAACCGCGACCTGCGGTTGCCACTTTGGTTAA(SEQ
ID NO：17).
Embodiments in accordance with the present invention, the NisB albumen is by SEQ ID NO：Nucleic acid sequence encoding shown in 10.That is,
Its sequence is as follows for nisB genes for the encoding gene of NisB albumen：
atgataaaaagttcatttaaagctcaaccgtttttagtaagaaatacaattttatctccaaacgataaacggagttttactgaatatactcaag
tcattgagactgtaagtaaaaataaagtttttttggaacagttactactagctaatcctaaactctatgatgttatgcagaaatataatgctggtctgtt
aaagaagaaaagggttaaaaaattatttgaatctatttacaagtattataagagaagttatttacgatcaactccatttggattatttagtgaaacttca
attggtgttttttcgaaaagttcacagtacaagttaatgggaaagactacaaagggtataagattggatactcagtggttgattcgcctagttcataa
aatggaagtagatttctcaaaaaagttatcatttactagaaataatgcaaattataagtttggagatcgagtttttcaagtttataccataaatagtagt
gagcttgaagaagtaaatattaaatatacgaatgtttatcaaattatttctgaattttgtgagaatgactatcaaaaatatgaagatatttgtgaaactg
taacgctttgctatggagacgaatatagagaactatcggaacaatatcttggcagtctgatagttaatcattatttgatctctaatttacaaaaagatt
tgttgtcagatttttcttggaacacttttttgactaaagttgaagcaatagatgaagataaaaaatatataattcctctgaaaaaagttcaaaagtttatt
caagaatactcagaaatagaaattggtgaaggtattgagaaactgaaagaaatatatcaggaaatgtcacaaattcttgagaatgataattatatt
caaattgatttaattagtgatagtgaaataaattttgatgttaaacaaaagcaacaattagaacatttagctgagtttataggaaatacgacaaaatct
gtaagaagaacatatttggatgactataaggataaatttatcgaaaaatatggtgtagatcaagaagtacaaataacagaattatttgattctacatt
tggcataggagctccatataattataatcatcctcgaaatgacttttatgagtccgaaccgagtactctatactattcagaagaggagagagaaaa
gtacctcagcatgtatgtagaagccgttaaaaatcataatgtaattaatcttgacgacttagagtctcattatcaaaaaatggacttagaaaagaaa
agtgaacttcaagggttagaattatttttgaatttggcaaaggagtatgaaaaagatatttttattttaggggatatcgttggaaataataatttggga
ggggcatcaggtagattttctgcactctctccggagttaacaagttatcatagaacgatagtagattctgtcgaaagagaaaatgagaataaaga
aattacatcgtgtgaaatagtatttcttccagaaaatatcagacatgctaacgttatgcatacatcaattatgaggaggaaagtacttccattttttac
aagtacaagtcacaatgaagttctgttaactaatatctatattggaatagacgaaaaagaaaaattttatgcacgagacatttcaactcaagaggta
ttgaaattctacattacaagcatgtacaataaaacgttattcagtaatgagctaagatttctttacgaaatttcattagatgacaagtttggtaatttacc
ttgggaacttatttacagagactttgattatattccacgtttagtatttgacgaaatagtaatatctcctgctaaatggaaaatttggggaagggatgt
aaatagtaagattacaataagagaacttattcaaagcaaagaaattcccaaagagttttatattgtcaatggagataataaagtttatttatcacagg
aaaacccattggatatggaaattttagagtcggcgataaagaagagctcaaaaagaaaagattttatagagctacaagaatattttgaagatgaa
aatatcataaataaaggacaaaaggggagagttgccgatgttgtagtgccttttattagaacgagagcattaggtaatgaagggagagcatttat
aagagagaaaagagtttcggttgaacggcgtgaaaaattgccctttaacgagtggctttatctcaagttgtacatttctataaatcgtcaaaatgaa
tttttactgtcgtatcttccagatattcagaaaatagtagcaaacctgggtggaaatctattcttcctaagatatactgatcctaaaccacatattagat
tgcgtataaaatgttcagatttatttttagcttacggatctattcttgaaatcttaaaaaggagtcggaaaaataggataatgtcaacttttgatatttct
atttatgatcaagaagtagaaagatatggtggatttgatactttagagttatccgaagcaatattttgtgccgattctaaaattattccaaatttgctta
cattgataaaagatactaataatgattggaaagtcgatgatgtatcaatcttggtgaattatttatatctgaaatgcttctttcagaatgataacaaaaa
gattcttaattttttgaatttagttagtcctaaaaaggttaaagaaaatgtcaatgaaaagattgaacattatcttaagcttctgaaagttaataatctag
gtgaccaaattttttatgacaagaattttaaagaattaaagcatgccataaaaaatttatttttaaaaatgatagctcaagattttgaacttcagaaagt
ttattcaattattgacagtatcattcatgtccataataaccgactaattggtattgaacgagataaagagaaattaatttattacacacttcaaaggttg
tttgtttcggaagaatacatgaaatga(SEQ ID NO：10).
Thereby, it is possible to efficiently express NisB albumen, and can effectively by the nisin using the NisB albumen
The specific serine and threonine of precursor peptide are dehydrated, and complete first step modification.
Embodiments in accordance with the present invention, the NisC albumen is by SEQ ID NO：Nucleic acid sequence encoding shown in 11.That is,
The encoding gene of NisC albumen is nisC genes, and its sequence is as follows：
atgaataaaaaaaatataaaaagaaatgttgaaaaaattattgctcaatgggatgagagaactagaaaaaataaagaaaacttcgatttc
ggagagttgactctctctacaggattgcctggtataattttaatgttagcggagttaaaaaataaagataactcaaagatatatcagaaaaagata
gacaattatattgaatatattgttagcaaactttcaacatatgggcttttaacaggatcactttattcgggagcagctggcattgcattaagtatccta
catttacgagaagatgacgaaaaatataagaatcttcttgatagcctaaatagatatatcgaatatttcgtcagagaaaaaattgaaggatttaattt
ggaaaacattactcctcctgattatgacgtgattgaaggtttatctgggatactttcctatctattattaatcaacgacgagcaatatgatgatttgaa
aatactcattatcaattttttatcaaatctgactaaagaaaacaaaggactaatatcgctttacatcaaatcggagaatcagatgtctcaatcagaaa
gtgagatgtatccactaggctgtttgaatatgggattagcacatggacttgctggagtgggctgtatcttagcttatgcccacataaaaggatata
gtaatgaagcctcgttgtcagctttgcaaaaaattatttttatttatgaaaagtttgaacttgaaaggaaaaaacagtttctatggaaagatggacttg
tagcagatgaattaaaaaaagagaaagtaattagggaagcaagtttcattagagatgcatggtgctatggaggtccaggtattagtctgctatac
ttatacggaggattagcactggataatgactattttgtagataaagcagaaaaaatattagagtcagctatgcaaaggaaacttggtattgattcat
atatgatttgccatggctattctggtttaatagaaatttgttctttatttaagcggctattaaatacaaaaaagtttgattcatacatggaagaatttaatg
ttaatagtgagcaaattcttgaagaatacggagatgaaagtggcacgggttttcttgaaggaataagtggctgtatactggtattatcgaaatttga
atattcaatcaattttacttattggagacaagcactgttactttttgacgattttttgaaaggagggaagaggaaatga(SEQ ID NO：11).
Thereby, it is possible to efficiently obtain NisC albumen, and the breast that can effectively make to be modified by NisB using the NisC albumen
Acid streptococci element precursor peptide forms wool sulphur ring, completes second step modification.
Embodiments in accordance with the present invention, are modified the nisin precursor peptide using NisB albumen and NisC albumen,
It is by least one realization selected from following manner：
(1) microorganism is made while expressing the nisin precursor peptide, NisB albumen and NisC albumen, so as to
The modification to the nisin precursor peptide is completed in the microbial body, before obtaining the nisin by modification
Body peptide；
(2) in making the microorganism while expressing the nisin precursor peptide and NisB albumen and NisC albumen
One of which, is then mixed the another kind in NisB albumen and NisC albumen with expression product, to obtain warp in vitro
Cross the nisin precursor peptide of modification；
(3) the NisB albumen, the NisC albumen are mixed in vitro with the nisin precursor peptide, with
Just the nisin precursor peptide by modification is obtained.
Thus, the modification to the nisin precursor peptide can be realized by above-mentioned various ways.
Embodiments in accordance with the present invention, the external enzymolysis is the albumen and the warp by being possible to recognize NisP cleavage sites
Crossing the nisin precursor peptide of modification carries out what external mixing was realized.Thus, microbial cell will not be damaged, and it is convenient fast
It is prompt, moreover it is possible to effectively to reduce production cost.
Embodiments in accordance with the present invention, the albumen for being capable of identify that NisP cleavage sites is NisP albumen, the NisP eggs
There is SEQ ID NO in vain：Amino acid sequence shown in 9.Specifically, the sequence of NisP albumen is as follows：
VKKILGFLFIVCSLGLSATVHGETTNSQQLLSNNINTELINHNSNAILSSTEGSTTDSINL
GAQSPAVKSTTRTELDVTGAAKTLLQTSAVQKEMKVSLQETQVSSEFSKRDSVTNKEAVP
VSKDELLEQSEVVVSTSSIQKNKILDNKKNRANFVTSSQLIKEKPSNSKDASGVIDNSASPL
SYRKAKEVVSLRQPLKNQKVEAQPLLISNSSEKKASVYTNSHDFWDYQWDMKYVTNNG
ESYALYQPSKKISVGIIDSGIMEEHPDLSNSLGNYFKNLVPKGGFDNEEPDETGNPSDIVDK
MGHGTEVAGQITANGNILGVAPGITVNIYRVFGENLSKSEWVARAIRRAADDGNKVINISA
GQYLMISGSYDDGTNDYQEYLNYKSAINYATAKGSIVVAALGNDSLNIQDNQTMINFLKR
FRSIKVPGKVVDAPSVFEDVIAVGGIDGYGNISDFSNIGADAIYAPAGTTANFKKYGQDKFV
SQGYYLKDWLFTTTNTGWYQYVYGNSFATPKVSGALALVVDKYGIKNPNQLKRFLLMNS
PEVNGNRVLNIVDLLNGKNKAFSLDTDKGQDDAINHKSMENLKESRDTMKQEQDKEIQR
NTNNNFSIKNDFHNISKEVISVDYNINQKMANNRNSRGTVSVRSQEILPVTGDGEDFLRAL
GIVCISILGILKRKTKN(SEQ ID NO：9).Thereby, it is possible to efficiently realize to the streptococcus lactis by modification
The external enzymolysis of plain precursor peptide, effectively obtains nisin.
Embodiments in accordance with the present invention, the NisP albumen is by SEQ ID NO：Nucleic acid sequence encoding shown in 18.Specifically,
The encoding gene of NisP albumen is NisP genes, and its sequence is as follows：
gtgaaaaaaatactaggtttcctttttatcgtttgttcgttgggtttatcagcaactgtgcatggggagacaacaaattcacaacagttactct
caaataatattaatacggaattaattaatcataattctaatgcaattttatcttcaacagagggatcaacgactgattcgattaatctaggggcgcag
tcacctgcagtaaaatcgacaacaaggactgaattggatgtaactggtgctgctaaaactttattacagacatcagctgttcaaaaagaaatgaa
agtttcgttgcaagaaactcaagttagttctgaattcagtaagagagatagcgttacaaataaagaagcagttccagtatctaaggatgagctact
tgagcaaagtgaagtagtcgtttcaacatcatcgattcaaaaaaataaaatcctcgataataagaagaatagagctaacttcgttacttcctctcag
cttattaaggaaaaaccatcaaattctaaagatgcatctggtgtaattgataattctgcttctcctctatcttatcgtaaagctaaggaagtggtatctc
ttagacaacctttaaaaaatcaaaaagtagaggcacaacctctattgataagtaattcttctgaaaagaaagcaagtgtttatacaaattcacatga
tttttgggattatcagtgggatatgaaatatgtgacaaataatggagaaagctatgcgctctaccagccctcaaagaaaatttctgttggaattattg
attcaggaatcatggaagaacatcctgatttgtcaaatagtttaggaaattattttaaaaatcttgttcctaagggagggtttgataatgaagaacct
gatgaaactggaaatccaagtgatattgtcgacaaaatgggacacgggacggaagtcgcaggtcagattacagcaaatggtaatattttagga
gtagcaccagggattactgtaaatatatacagagtatttggtgaaaatctttcgaaatcggaatgggtagctagagcaataagaagagctgcgg
atgatgggaacaaggtcatcaatataagtgctggacagtatcttatgatttcaggatcgtatgatgatggaacaaatgattatcaagagtatcttaa
ttataagtcagcaataaattatgcaacagcaaaaggaagtattgttgtcgcagctcttggtaatgatagtttaaacatacaagataaccaaacaat
gataaactttcttaagcgtttcagaagtataaaggttcctggaaaagttgtagatgcaccgagtgtatttgaggatgtaatagccgtaggtggaat
agatggttatggtaatatttctgattttagtaatattggagcggatgcaatttatgctcctgctggcacaacggccaattttaaaaaatatgggcaag
ataaatttgtcagtcagggttattatttgaaagattggctttttacaactactaatactggctggtaccaatatgtttatggcaactcatttgctactcct
aaagtatctggggcactggcattagtagttgataaatatggaataaagaatcctaaccaactaaaaaggtttcttctaatgaattctccagaagtta
atgggaatagagtattgaatattgttgatttattgaatgggaaaaataaagcttttagcttagatacagataaaggtcaggatgatgctattaaccat
aaatcaatggagaatcttaaagagtctagggatacaatgaaacaggaacaagataaagaaattcaaagaaatacaaataacaatttttctatcaa
aaatgattttcataacatttcaaaagaagtaatttcagttgattataatattaatcaaaaaatggctaataatcgaaattcgagaggtactgtttctgta
cgaagtcaagaaattttacctgttactggagatggagaagattttttacgggctttaggtatagtgtgtatctcaatccttggtatattgaaaagaaa
gactaaaaattga(SEQ ID NO：18).
Embodiments in accordance with the present invention, the albumen for being capable of identify that NisP cleavage sites is trypsase, preferably Trypsin.
Thereby, it is possible to efficiently realize the external enzymolysis to the nisin precursor peptide by modification, streptococcus lactis is effectively obtained
Element, and trypsase obtains easy, is easy to the industrialization and large-scale promotion of the inventive method.
Embodiments in accordance with the present invention, the microorganism does not possess natural nisin route of synthesis.Because, tool
The microorganism of standby natural nisin route of synthesis, the nisin of its natural synthesis can also destroy own cells and break
Split, influence the implementation of the method for the present invention.Some preferred embodiments of the invention, the microorganism be selected from Escherichia coli,
At least one of trichoderma, yeast, streptomycete, bacillus subtilis and aspergillus oryzae.
Embodiments in accordance with the present invention, using selected from Escherichia coli, trichoderma, yeast, streptomycete, bacillus subtilis and meter Qu
At least one expression of mould obtains the NisB albumen.Thereby, it is possible to efficiently produce NisB albumen.
Embodiments in accordance with the present invention, using selected from Escherichia coli, trichoderma, yeast, streptomycete, bacillus subtilis and meter Qu
At least one expression of mould obtains the NisC albumen.Thereby, it is possible to efficiently produce NisC albumen.
Embodiments in accordance with the present invention, using selected from Escherichia coli, trichoderma, yeast, streptomycete, bacillus subtilis and meter Qu
At least one expression of mould obtains the NisP albumen.Thus, NisP protein yields are high, and quality is good, can be effective for
Above-mentioned external enzymolysis.
Additional aspect of the invention and advantage will be set forth in part in the description, and partly will become apparent from the description below,
Or recognized by practice of the invention.
Brief description of the drawings
Of the invention above-mentioned and/or additional aspect and advantage be will be apparent from description of the accompanying drawings below to embodiment is combined and
It is readily appreciated that, wherein：
Fig. 1 shows the structural representation of plasmid pRL300 according to an embodiment of the invention；
Fig. 2 shows the structural representation of plasmid pRL301 according to an embodiment of the invention；
Fig. 3 shows the structural representation of plasmid pRL302 according to an embodiment of the invention；
Fig. 4 shows the structural representation of plasmid pRL303 according to an embodiment of the invention；
Fig. 5 shows the structural representation of plasmid pRL304 according to an embodiment of the invention；
Fig. 6 shows the structural representation of pRL305 plasmids according to an embodiment of the invention；
Fig. 7 shows the structural representation of pRL306 plasmids according to an embodiment of the invention；
Fig. 8 shows the structural representation of pRL307 plasmids according to an embodiment of the invention；
Fig. 9 shows the structural representation of pRL308 plasmids according to an embodiment of the invention；
Figure 10 shows the structural representation of pRL309 plasmids according to an embodiment of the invention；
Figure 11 shows the structural representation of pRL310 plasmids according to an embodiment of the invention；
Figure 12 shows the structural representation of pRL311 plasmids according to an embodiment of the invention；
Figure 13 shows the structural representation of plasmid pRL312 according to an embodiment of the invention
Figure 14 shows the structural representation of plasmid pRL313 according to an embodiment of the invention；
Figure 15 shows the structural representation of plasmid pRL314 according to an embodiment of the invention；
Figure 16 shows the structural representation of plasmid pRL315 according to an embodiment of the invention；
Figure 17 shows the structural representation of plasmid pRL316 according to an embodiment of the invention；And
Figure 18 shows the structural representation of plasmid pRL316 according to an embodiment of the invention.
Specific embodiment
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that the following examples
The present invention is merely to illustrate, and be should not be taken as limiting the scope of the invention.Unreceipted particular technique or condition, press in embodiment
According to the technology or condition described by document in the art, (for example reference J. Pehanorm Brookers etc. write, what Huang Peitang etc. was translated《Molecule
Cloning experimentation guide》, the third edition, Science Press) or carried out according to product description.Agents useful for same or instrument are unreceipted
Production firm person, be can by city available from conventional products, can for example purchase from Sigma companies.
Embodiment 1 prepares nisin
First, semi-matured Nisin precursor peptides are synthesized
According to following steps, synthesize semi-matured in yeast, streptomycete, bacillus subtilis, aspergillus oryzae and trichoderma respectively
Nisin precursor peptides：
1st, semi-matured Nisin precursor peptides are synthesized in saccharomyces cerevisiae
GAL7, GAL10 and GAL1 promoter are added respectively in nisA, nisB and nisC gene front end, and at these three
Terminator is added behind gene respectively, then these three expression cassettes are cloned in the saccharomyces cerevisiae expression of removal original promoter
On pYes2, plasmid pRL300, plasmid figure such as Fig. 1 are formed.The conversion of this plasmid is entered in saccharomyces cerevisiae INVSC1, is obtained
To saccharomyces cerevisiae expression bacterium RL300.
(contain 2% glucose) from picking RL300 monoclonals to 5mL SC-U Selective agar mediums on selection flat board, 30 DEG C are shaken
Bottle overnight after, (containing 2% glucose) in being transferred to 50mL SC-U Selective agar mediums, 30 DEG C of Shaking cultures are after 6 hours,
The OD values of culture measurement overnight culture.Calculating meets bacterium solution amounts of the 300mL inducing cultures OD for needed for 0.4.Calculate
Method is as follows：The bacterium solution OD of incubated overnight is a.Bacterium solution amount required for 300mL Selective agar mediums is (120/a) mL.Take
State the desired amount of bacterium solution of step, 4 DEG C, 1500rpm be centrifuged 5 minutes, collects thalline.With 1-2mL inducing cultures (SC-U,
Containing 2% raffinose) resuspended thalline, in addition 300mL inducing cultures (SC-U, containing 2% raffinose), 30 DEG C of shaking flasks
Culture 1-2 hours, adds 1% galactolipin to be induced, the collects thalline after 24 hours after induction, then by after bacterial cell disruption
Obtain immature Nisin albumen.
2nd, semi-matured Nisin precursor peptides are synthesized in streptomycete
RBS sequences will be added by design of primers between nisA and nisB, two terminal sequences is expanded together by PCR,
RBS sequences and nisC sequences are being added below again simultaneously, this whole section of sequence is being cloned on carrier pIB139, constituting chain
Mould expression vector pRL301, plasmid figure such as Fig. 2.This plasmid is thin into streptomycete FR008 host by Conjugative tiansfer
In born of the same parents, streptomycete expression bacterium RL301. is formed
From selection flat board in successful RL301 to the 5mL TSBY culture mediums of picking empirical tests, 28 DEG C of shaking flasks overnight after, will
It is transferred in 50mL TSBY culture mediums, 28 DEG C continue Shaking cultures after bacterium it is long it is dense after be transferred to according still further to 2% switching amount
Cultivated 36 hours in the TSBY culture mediums of 300mL, collects thalline, then immature Nisin eggs will be obtained after bacterial cell disruption
In vain.
3rd, semi-matured Nisin precursor peptides are synthesized in bacillus subtilis
The genetic fragment of nisA, nisB, nisC coexpression is expanded from pRL301, bacillus subtilis expression is cloned in
On carrier pHT43, bacillus subtilis expression vector pRL302, plasmid figure such as Fig. 3 are constituted.This plasmid is converted and is entered
In bacillus subtilis WB800N, bacillus subtilis expression bacterium RL302. is formed
The RL302 inoculations of picking good authentication contain in 10ug/mL LB culture mediums in 5mL, are placed in shaking table 37
Degree incubated overnight, is inoculated in LBs of the 500mL containing antibiotic and trains respectively by 1% inoculum concentration by above-mentioned one-level nutrient solution overnight
Support in base, 37 degree of cultures to OD reach 0.8 or so, add the IPTG inductions of final concentration of 1mM, temperature is reduced to 28 degree.
Extracted from zymotic fluid within 24 hours after induction and obtain immature Nisin albumen.
4th, semi-matured Nisin precursor peptides are synthesized in aspergillus oryzae expression bacterial strain RL303
The gpdA promoters from aspergillus nidulans are added respectively before tri- genes of nisA, nisB and nisC, from aspergillus niger
PkiA promoters and the trpC promoters from aspergillus nidulans, and respectively plus aspergillus nidulans trpC terminators, aspergillus oryzae
AgdA terminators and aspergillus niger glaA terminators, while cloning aspergillus nidulans from the plasmid pYH-WA-pyrG-KI
This four fragments are joined end to end rear clone to forming plasmid pRL303, plasmid on pMD-18T carriers by pyrG genetic fragments
Figure such as Fig. 4.It is transferred to after plasmid enzyme restriction is linearized in aspergillus oryzae nutritional deficiency type bacterial strain (pyrG-), obtains aspergillus oryzae expression
Bacterial strain RL303.
By in RL303 inoculations to PDB culture mediums, 28 DEG C, 220rpm is cultivated 3-4 days, thalline is collected by filtration and extracts egg
In vain.
5th, semi-matured Nisin precursor peptides are synthesized in trichoderma reesei
Respectively plus trichoderma reesei (Trichoderma reesei) cbhI genes before tri- genes of nisA, nisB and nisC
Promoter, the promoter and microassembly robot (Penicillium of long shoot trichoderma (Trichoderma longibrachiatum) egl1 genes
Janthinellum) the promoter of egl2 genes, and the trpC plus aspergillus nidulans (Aspergillus nidulans) terminates respectively
Son and Nos terminators, these three expression cassettes are cloned into the pCambia1301 for having changed hygromycin gene promoter
On, obtain plasmid pRL311 (see Figure 12).Plasmid pRL311 is transformed into Agrobacterium tumefaciems EHA105, by agriculture bar
The genetic transforming method of bacterium mediation converts trichoderma reesei (Trichoderma reesei), obtains recombinant bacterial strain RL311.
This recombinant bacterial strain RL311 is inoculated into the glucose containing 5g/L, the fibrous matter of 10g/L and the microcrystalline cellulose of 10g/L
30mL basal fermentation mediums (BFM) culture medium in, the shaking flask of 250mL, 200rpm, 28 DEG C, pH5-6, vibration
Culture 5d, pH is adjusted using 10% ammoniacal liquor, and albumen is collected after fermentation ends.
2nd, expression and purification NisP albumen
Using e. coli protein expression and purification system, nisP genes are cloned in pET28 (a) by NdeI and XhoI sites
On plasmid, plasmid pRL304, plasmid figure such as Fig. 5 are formed.The plasmid is imported large intestine is formed in e. coli bl21 (DE3)
Bacillus expression bacterium RL304.
By RL304 in 37 DEG C of the 5mL LB culture mediums containing kanamycins, 200rpm is cultivated 12 hours, by 1% inoculation
Amount transfers during 300mL contains kanamycins LB culture mediums Escherichia coli overnight culture, 37 DEG C, 200rpm shaking tables
Culture adds 0.25mM IPTG to be induced in about 2 hours to OD values about 0.6, and temperature is reduced to 18 DEG C of continuation after induction
Collects thalline after cultivating 18 hours.Thalline is suspended in Tris buffer solutions by after ultrasonication, supernatant being collected, by supernatant
By nickel post, NisP albumen can be combined on nickel post, finally again with the elution buffer containing imidazoles by NisP albumen from nickel post
On elute, as purify the NisP albumen for obtaining.
3rd, Nisin precursor peptides are synthesized：NisA albumen, NisZ albumen, NisQ albumen, NisF albumen, NisU albumen and
NisU2 albumen
(1) NisA albumen is synthesized
According to following steps, synthesize NisA albumen in yeast, streptomycete, bacillus subtilis and aspergillus oryzae respectively：
1st, NisA albumen is synthesized in saccharomyces cerevisiae
NisA is cloned on saccharomyces cerevisiae expression pYes2, plasmid pRL305, plasmid figure such as Fig. 6 is formed.By this
Plasmid conversion enters in saccharomyces cerevisiae INVSC1, obtains saccharomyces cerevisiae expression bacterium RL305.
(contain 2% glucose) from picking RL305 monoclonals to 5mL SC-U Selective agar mediums on selection flat board, 30 DEG C are shaken
Bottle overnight after, (containing 2% glucose) in being transferred to 50mL SC-U Selective agar mediums, 30 DEG C of Shaking cultures are after 6 hours,
The OD values of culture measurement overnight culture.Calculating meets bacterium solution amounts of the 300mL inducing cultures OD for needed for 0.4.Calculate
Method is as follows：The bacterium solution OD of incubated overnight is a.Bacterium solution amount required for 300mL Selective agar mediums is (120/a) mL.Take
The desired amount of bacterium solution of step is stated, 4 DEG C of 1500rpm are centrifuged 5 minutes, collects thalline.With 1-2mL inducing cultures (SC-U,
Containing 2% raffinose) resuspended thalline, in addition 300mL inducing cultures (SC-U, containing 2% raffinose), 30 DEG C of shaking flasks
Culture 1-2 hours, adds 1% galactolipin to be induced, and collects thalline after 24 hours, therefrom obtains NisA after induction
Albumen.
2nd, NisA albumen is synthesized in streptomycete
By nisA gene clonings on carrier pIB139, streptomyces expression vector pRL306, plasmid figure such as Fig. 7 are constituted.By this
Individual plasmid is entered in streptomycete FR008 host cells by Conjugative tiansfer, forms streptomycete expression bacterium RL306.
From selection flat board in successful RL306 to the 5mL TSBY culture mediums of picking empirical tests, 28 DEG C of shaking flasks overnight after, will
It is transferred in 50mL TSBY culture mediums, 28 DEG C continue Shaking cultures after bacterium it is long it is dense after be transferred to according still further to 2% switching amount
Cultivated 36 hours in the TSBY culture mediums of 300mL, collects thalline, then NisA albumen will be obtained after bacterial cell disruption.
3rd, NisA albumen is synthesized in bacillus subtilis
By nisA gene clonings on bacillus subtilis expression vector pHT43, bacillus subtilis expression vector is constituted
PRL307, plasmid figure such as Fig. 8.The conversion of this plasmid is entered in bacillus subtilis WB800N, bacillus subtilis are formed
Bacterium expression bacterium RL307.
The RL307 inoculations of picking good authentication contain in 10ug/mL LB culture mediums in 5mL, are placed in shaking table 37
Degree incubated overnight, is inoculated in LBs of the 500mL containing antibiotic and trains respectively by 1% inoculum concentration by above-mentioned one-level nutrient solution overnight
Support in base, 37 degree of cultures to OD reach 0.8 or so, add the IPTG inductions of final concentration of 1mM, temperature is reduced to 28 degree.
Extracted from zymotic fluid within 24 hours after induction and obtain NisA albumen.
4th, NisA albumen is synthesized in aspergillus oryzae
Respectively plus gpdA promoters and trpC terminators from aspergillus nidulans before and after nisA genes, while from plasmid
The pyrG genetic fragments of aspergillus nidulans are cloned on pYH-WA-pyrG-KI, this four fragments rear clone that joins end to end is arrived
Plasmid pRL308, plasmid figure such as Fig. 9 are formed on pMD-18T carriers.Aspergillus oryzae nutrition is transferred to after plasmid enzyme restriction is linearized
In deletion form bacterial strain (pyrG-), aspergillus oryzae expression bacterial strain RL308 is obtained.
By in RL308 inoculations to PDB culture mediums, 28 DEG C, 220rpm is cultivated 3-4 days, thalline is collected by filtration and extracts egg
White NisA.
5th, NisA albumen is synthesized in Pichia pastoris
NisA is cloned on pGAPZ- α plasmids, Gene A is between α-signal peptide and AOX1 terminators, utilized
Composing type GAP promoters are expressed, and obtain plasmid pRL312, plasmid figure such as Figure 13.PRL312 is transformed into complete red ferment
In female GS115, screening obtains recombinant bacterial strain RL312.
Recombinant bacterial strain RL312 is inoculated into YPD culture mediums, 30 DEG C, after 220rpm is cultivated 96 hours, collects zymotic fluid
Extract albumen NisA.
(2) NisZ albumen, NisQ albumen, NisF albumen, NisU albumen and NisU2 albumen are respectively synthesized
By nisZ gene clonings on pET28 (a) plasmids, plasmid pRL313, plasmid figure such as Figure 14 are formed.The plasmid is imported
Bacillus coli expression bacterium RL313 is formed in e. coli bl21 (DE3).
By nisQ gene clonings on pET28 (a) plasmids, plasmid pRL314, plasmid figure such as Figure 15 are formed.The plasmid is led
Enter formation Bacillus coli expression bacterium RL314 in e. coli bl21 (DE3).
By nisF gene clonings on pET28 (a) plasmids, plasmid pRL315, plasmid figure such as Figure 16 are formed.The plasmid is imported
Bacillus coli expression bacterium RL315 is formed in e. coli bl21 (DE3).
By nisU gene clonings on pET28 (a) plasmids, plasmid pRL316, plasmid figure such as Figure 17 are formed.The plasmid is led
Enter formation Bacillus coli expression bacterium RL316 in e. coli bl21 (DE3).
By nisU2 gene clonings on pET28 (a) plasmids, plasmid pRL317, plasmid figure such as Figure 18 are formed.The plasmid is led
Enter formation Bacillus coli expression bacterium RL317 in e. coli bl21 (DE3).
Respectively by bacterial strain RL313, RL314, RL315, RL316, RL317 in the 5mL LB cultures containing kanamycins
37 DEG C of base, 200rpm is cultivated 12 hours, Escherichia coli overnight culture is transferred by 1% inoculum concentration is contained into 300mL
In kanamycins LB culture mediums, 37 DEG C, 200rpm shaking table cultures add 0.25mM in about 2 hours to OD values about 0.6
IPTG is induced, and temperature is reduced to collects thalline after 18 DEG C of continuation are cultivated 18 hours after induction.Be can obtain after broken corresponding
Albumen.
4th, expression and purification NisB albumen
Using e. coli protein expression and purification system, by nisB gene clonings on pET28 (a) plasmids, plasmid pRL309 is formed,
Plasmid figure such as Figure 10.The plasmid is imported Bacillus coli expression bacterium RL309 is formed in e. coli bl21 (DE3).
By RL309 in 37 DEG C of the 5mL LB culture mediums containing kanamycins, 200rpm is cultivated 12 hours, by 1% inoculation
Amount transfers during 300mL contains kanamycins LB culture mediums Escherichia coli overnight culture, 37 DEG C, the training of 200rpm shaking tables
Support about 2 hours to OD values about 0.6, add 0.25mM IPTG to be induced, temperature is reduced to 18 DEG C and continues to train after induction
Collects thalline after supporting 18 hours.Thalline is suspended in Tris buffer solutions by after ultrasonication, collecting supernatant, will be upper clear and coherent
Cross nickel post, NisB albumen can be combined on nickel post, finally again with the elution buffer containing imidazoles by NisB albumen from nickel post
Elute, as purify the NisB albumen for obtaining.
5th, expression and purification NisC albumen
Using e. coli protein expression and purification system, by nisC gene clonings on pET28 (a) plasmids, plasmid pRL310 is formed,
Plasmid figure such as Figure 11.The plasmid is imported Bacillus coli expression bacterium RL310 is formed in e. coli bl21 (DE3).
By RL310 in 37 DEG C of the 5mL LB culture mediums containing kanamycins, 200rpm is cultivated 12 hours, by 1% inoculation
Amount transfers during 300mL contains kanamycins LB culture mediums Escherichia coli overnight culture, 37 DEG C, 200rpm shaking tables
Culture adds 0.25mM IPTG to be induced in about 2 hours to OD values about 0.6, and temperature is reduced to 18 DEG C of continuation after induction
Collects thalline after cultivating 18 hours.Thalline is suspended in Tris buffer solutions by after ultrasonication, supernatant being collected, by supernatant
By nickel post, NisC albumen can be combined on nickel post, finally again with the elution buffer containing imidazoles by NisC albumen from nickel post
On elute, as purify the NisC albumen for obtaining.
6th, with the NisP semi-matured Nisin precursor peptides of albumen vitro enzyme solution
For several semi-matured Nisin precursor peptides being respectively synthesized in different microorganisms in step one, respectively carry out following
Operation：
Suspended semi-matured Nisin precursor peptides using 0.5M phosphate buffers, and add the NisP eggs that acquisition is purified in step 2
In vain, then after 30 degrees Celsius of lower lucifuges are reacted 5 hours, collecting product carries out Mass Spectrometric Identification.As a result, maturation has been obtained
Nisin albumen.
7th, semi-matured Nisin precursor peptides are digested in vitro with trypsase Trypsin
For several semi-matured Nisin precursor peptides being respectively synthesized in different microorganisms in step one, respectively carry out following
Operation：
Semi-matured Nisin precursor peptides are suspended with 0.5M phosphate buffers, Trypsin is added and in 30 degrees Celsius of lower lucifuges
After reaction 5 hours, collecting product carries out Mass Spectrometric Identification.As a result, the Nisin albumen of maturation has been obtained.
8th, with NisB, NisC and NisP albumen vitro enzyme solution Nisin precursor peptides
For be respectively synthesized in different microorganisms in step 3 several Nisin precursor peptides (i.e. NisA albumen, NisZ albumen,
NisQ albumen, NisF albumen, NisU albumen and NisU2 albumen), respectively carry out following operation：
Nisin precursor peptides are suspended with 0.5M phosphate buffers, NisB albumen, the step 5 obtained in addition purification step four
The NisP albumen obtained in the NisC albumen and step 2 of middle acquisition, and after 30 degrees Celsius of lower lucifuges are reacted 10 hours,
Collecting product carries out Mass Spectrometric Identification.As a result, the Nisin albumen of maturation has been obtained.
9th, Nisin protein yields and quality testing
Reference literature Aeration and fermentation strategies on nisin production.Biotechnology Letters.
The detection method reported in 2015, olume 37, Issue 10, pp 2039-2045, Nisin albumen in detecting step six, seven, eight
Yield, as a result find, the utilization lactic acid bacteria that the yield of the Nisin of acquisition is all remarkably higher than current report produces the product of Nisin
Amount.Document Aeration and fermentation strategies on nisin production.Biotechnology as escribed above
Nisin yield in Letters.2015, olume 37, Issue 10, pp 2039-2045 is 15400IU/mL, and the present invention is above-mentioned
The yield of Nisin albumen is significantly higher than 15400IU/mL in step 6, seven, eight.Thus, show the present invention relative to existing
Technology can produce more Nisin, namely yield is higher.
Found by mass spectral analysis, the purity of the Nisin albumen obtained in above-mentioned steps of the present invention six, seven, eight is above at present
Commercially available Nisin application products (not including standard items).
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specific example ",
Or the description of " some examples " etc. means to combine specific features, structure, material or feature bag that the embodiment or example are described
It is contained at least one embodiment of the invention or example.In this manual, the schematic representation to above-mentioned term not necessarily refers to
Be identical embodiment or example.And, the specific features of description, structure, material or feature can be at any one
Or combined in an appropriate manner in multiple embodiments or example.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：This is not being departed from
Can these embodiments be carried out with various changes, modification, replacement and modification in the case of the principle and objective of invention, it is of the invention
Scope is limited by claim and its equivalent.
Claims (11)
1. a kind of method for preparing nisin, it is characterised in that comprise the following steps：
Nisin precursor peptide is obtained using microbial expression；
The nisin precursor peptide is modified using NisB albumen and NisC albumen, to obtain the nisin precursor peptide by modifying；And
The nisin precursor peptide by modification is digested in vitro using the albumen for being capable of identify that NisP cleavage sites, to obtain nisin,
Wherein, the NisB albumen has SEQ ID NO：Amino acid sequence shown in 1, the NisC albumen has SEQ ID NO：Amino acid sequence shown in 2.
2. method according to claim 1, it is characterized in that, the nisin precursor peptide be selected from least one of NisA albumen, NisZ albumen, NisQ albumen, NisF albumen, NisU albumen and NisU2 albumen, wherein, the NisA albumen has SEQ ID NO：Amino acid sequence shown in 3, the NisZ albumen has SEQ ID NO：Amino acid sequence shown in 4, the NisQ albumen has SEQ ID NO：Amino acid sequence shown in 5, the NisF albumen has SEQ ID NO：Amino acid sequence shown in 6, the NisU albumen has SEQ ID NO：Amino acid sequence shown in 7, the NisU2 albumen has SEQ ID NO：Amino acid sequence shown in 8,
Optionally, the NisA albumen is by SEQ ID NO：Nucleic acid sequence encoding shown in 12, the NisZ albumen is by SEQ ID NO：Nucleic acid sequence encoding shown in 13, the NisQ albumen is by SEQ ID NO：Nucleic acid sequence encoding shown in 14, the NisF albumen is by SEQ ID NO：Nucleic acid sequence encoding shown in 15, the NisU albumen is by SEQ ID NO：Nucleic acid sequence encoding shown in 16, the NisU2 albumen is by SEQ ID NO：Nucleic acid sequence encoding shown in 17,
Optionally, the NisB albumen is by SEQ ID NO：Nucleic acid sequence encoding shown in 10,
Optionally, the NisC albumen is by SEQ ID NO：Nucleic acid sequence encoding shown in 11.
3. method according to claim 1, it is characterised in that the nisin precursor peptide is modified using NisB albumen and NisC albumen, is by least one realization selected from following manner：
(1) make the microorganism while expressing the nisin precursor peptide, NisB albumen and NisC albumen, to complete the modification to the nisin precursor peptide in the microbial body, obtain the nisin precursor peptide by modification；
(2) microorganism is made while expressing the one of which in the nisin precursor peptide and NisB albumen and NisC albumen, then the another kind in NisB albumen and NisC albumen is mixed in vitro with expression product, to obtain the nisin precursor peptide by modifying；
(3) the NisB albumen, the NisC albumen are mixed in vitro with the nisin precursor peptide, to obtain the nisin precursor peptide by modifying.
4. method according to claim 1, it is characterised in that the external enzymolysis is to carry out mix realization in vitro by being possible to recognize the albumen and the nisin precursor peptide by modification of NisP cleavage sites.
5. method according to claim 1, it is characterised in that the albumen for being capable of identify that NisP cleavage sites is NisP albumen, the NisP albumen has SEQ ID NO：Amino acid sequence shown in 9,
Optionally, the NisP albumen is by SEQ ID NO：Nucleic acid sequence encoding shown in 18.
6. method according to claim 1, it is characterised in that the albumen for being capable of identify that NisP cleavage sites is trypsase, preferably Trypsin.
7. method according to claim 1, it is characterised in that the microorganism does not possess natural nisin route of synthesis.
8. method according to claim 7, it is characterised in that the microorganism is at least one selected from Escherichia coli, trichoderma, yeast, streptomycete, bacillus subtilis and aspergillus oryzae.
9. method according to claim 1, it is characterised in that obtain the NisB albumen using at least one expression selected from Escherichia coli, trichoderma, yeast, streptomycete, bacillus subtilis and aspergillus oryzae.
10. method according to claim 1, it is characterised in that obtain the NisC albumen using at least one expression selected from Escherichia coli, trichoderma, yeast, streptomycete, bacillus subtilis and aspergillus oryzae.
11. methods according to claim 1, it is characterised in that obtain the NisP albumen using at least one expression selected from Escherichia coli, trichoderma, yeast, streptomycete, bacillus subtilis and aspergillus oryzae.
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Cited By (4)
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| CN110627880A (en) * | 2018-06-25 | 2019-12-31 | 武汉臻智生物科技有限公司 | Isolated polypeptides and uses thereof |
| CN114250189A (en) * | 2021-12-08 | 2022-03-29 | 江南大学 | Heterologous expression system for synthesizing Nisin and application thereof |
| CN114717254A (en) * | 2022-04-29 | 2022-07-08 | 深圳大学 | Method for displaying and expressing Nisin by using yeast engineering bacteria and application of Nisin |
| CN114908114A (en) * | 2022-04-29 | 2022-08-16 | 深圳大学 | Yeast cell gene modification method, recombinant yeast and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110627880A (en) * | 2018-06-25 | 2019-12-31 | 武汉臻智生物科技有限公司 | Isolated polypeptides and uses thereof |
| CN110627880B (en) * | 2018-06-25 | 2024-03-19 | 武汉合生科技有限公司 | Isolated polypeptides and uses thereof |
| CN114250189A (en) * | 2021-12-08 | 2022-03-29 | 江南大学 | Heterologous expression system for synthesizing Nisin and application thereof |
| CN114717254A (en) * | 2022-04-29 | 2022-07-08 | 深圳大学 | Method for displaying and expressing Nisin by using yeast engineering bacteria and application of Nisin |
| CN114908114A (en) * | 2022-04-29 | 2022-08-16 | 深圳大学 | Yeast cell gene modification method, recombinant yeast and application thereof |
| CN114717254B (en) * | 2022-04-29 | 2024-03-12 | 深圳大学 | Method for displaying and expressing Nisin by yeast engineering bacteria and application thereof |
| CN114908114B (en) * | 2022-04-29 | 2024-03-12 | 深圳大学 | Yeast cell gene modification method, recombinant yeast and application thereof |
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