Polypeptide, its polynucleotides of separation, and the additive comprising polypeptide, its purposes and method
阅读说明：本技术 多肽，分离的其多核苷酸，以及包含多肽的添加剂、其用途及方法 (Polypeptide, its polynucleotides of separation, and the additive comprising polypeptide, its purposes and method ) 是由 S·弗鲁豪夫 M·萨姆海塞尔 M·费菲尔 D·摩尔 G·沙特兹迈尔 E·M·宾德 于 2014-08-27 设计创作，主要内容包括：以水解方式裂解玉米赤霉烯酮和/或至少一种玉米赤霉烯酮衍生物的多肽,其是具有选自SEQ ID No.1-15的氨基酸序列或其功能性变体的水解酶,其中在所述功能性变体与至少一个所述氨基酸序列之间的序列同一性为至少40％；以及包含所述多肽的添加剂；以及编码所述多肽的分离的多核苷酸；以及用于用所述多肽来以水解方式裂解玉米赤霉烯酮和/或至少一种玉米赤霉烯酮衍生物的方法。(The polypeptide of zearalenone and/or at least one zearalenone derivative is cracked with hydrolysis method, it is the hydrolase with amino acid sequence or its functional variant thereof selected from SEQ ID No.1-15, wherein the sequence identity between the functional variant thereof and at least one described amino acid sequence is at least 40%；And the additive comprising the polypeptide；And the isolated polynucleotides of coding said polypeptide；And the method for cracking zearalenone and/or at least one zearalenone derivative with the polypeptide with hydrolysis method.)
1. cracking the polypeptide of zearalenone and/or at least one zearalenone derivative, the jade with hydrolysis method Zearlenone derivative is selected from α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S and ZAN, which is characterized in that described more Peptide is the amino acid sequence with SEQ ID No.11 or the hydrolase of its functional variant thereof, wherein the functional variant thereof with Sequence identity between the amino acid sequence is at least 83%,
Wherein the polypeptide includes at least one conserved amino acid sequence section or its functional variant thereof, wherein the amino acid sequence The functional variant thereof of column section has at least 84% sequence identity, and at least one described conserved amino acid sequence section Amino acid sequence+89 to+145 or+223 to+228 selected from the sequence with SEQ ID No.1, and
Wherein the polypeptide has at least specific activity of 0.01U/mg.
2. polypeptide according to claim 1, which is characterized in that the polypeptide include at least one conserved amino acid sequence section or Its functional variant thereof, wherein the functional variant thereof of the amino acid sequence segment has at least 92% sequence identity, and At least one described conserved amino acid sequence section is the amino acid sequence+223 to+228 of the sequence with SEQ ID No.1.
3. polypeptide according to claim 2, which is characterized in that the functional variant thereof of the amino acid sequence segment has at least 98% sequence identity.
4. according to claim 1 to one of 3 polypeptide, which is characterized in that the polypeptide at least one be selected from following position Locate at least one mutation for the amino acid sequence for having about SEQ ID No.1: 22,23,25,26,27,29,31,32,35, 37、42、43、46、51、53、54、57、60、69、72、73、78、80、84、88、95、97、99、114、118、119、123、132、 141、146、148、149、154、163、164、165、169、170、172、176、180、182、183、190、191、194、196、 197、198、201、204、205、206、207、208、209、210、212、213、214、216、217、220、221、222、229、 231、233、238、240、244、245、246、248、249、251、254、256、260、262、263、266、269、271、277、 280、281、282、283、284、285、286、287、292、296、298、302、307、308、309、311、314、317、319、 321,323,325 and 326.
5. according to claim 1 to one of 3 polypeptide, which is characterized in that the polypeptide has selected from following about SEQ ID The amino acid sequence of No.1 at least one mutation: D22A, S23Q, S23L, N25D, I26V, F27Y, F27H, S29P, R31A, F32Y、R35K、R35Q、V37A、V42I、V43T、F46Y、S51E、S51D、D53G、N54M、N54R、L57V、L60I、S69G、 P72E、V73A、A78S、N80H、F84Y、I88L、T95S、T97A、R99K、I114M、I118V、K119R、V123I、L132V、 A141S、I146V、I146L、A148G、A149V、A154P、P163T、A164T、Y165C、Y165H、V169I、L170R、 A172G、A176M、A176V、Y180F、D182T、F183Y、I190V、G191S、K194T、K194E、F196Y、V197C、 V197R、E198R、E198S、K201D、K201G、P204S、P204A、A205S、K206P、A207M、M208A、Q209R、 L210A、L210S、ΔP212、T213V、P214A、E216T、E216G、A217I、N220H、L221M、K222R、K222Q、 G229A、A231V、F233W、F233Y、F233H、A238G、H240N、H240S、D244E、R245Q、M246L、S248T、 S248N、S248G、Q249R、K251N、I254V、I256L、A260M、T262D、T262G、I263T、E266D、E269H、 E269N、L271V、L277E、E280A、E280L、H281R、H281Q、A282V、Q283R、D284L、D284R、I285L、 I286M、R287E、R287D、R292K、R292T、Q296A、Q296E、H298V、L302S、L307Q、F308S、D309A、 A311P、A314V、L317F、S319Q、S319P、S319R、S321A、S321T、T323A、P325A、A326P。
6. isolated polynucleotides have the nucleotide sequence of coding polypeptide, wherein the polypeptide has hydrolysed corn red mould The characteristic of ketenes and/or at least one zearalenone derivative, the zearalenone derivative are selected from α-ZEL, β- ZEL, α-ZAL, β-ZAL, Z14G, Z14S and ZAN, which is characterized in that the nucleotide sequence coded at least one is according to right It is required that one of 1 to 5 polypeptide.
7. cracking zearalenone with hydrolysis method and/or at least one zearalenone derivative being used for generate The additive of the feed of pig, poultry or aquaculture, the additive is for being added to food or distiller's dried grain, the Gibberella zeae Ketenes derivative is selected from α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S and ZAN, which is characterized in that the additive packet Polypeptide containing amino acid sequence or its functional variant thereof with SEQ ID No.11, wherein the functional variant thereof with it is described Sequence identity between amino acid sequence is at least 83%.
8. additive according to claim 7, which is characterized in that the additive further includes auxiliary agent.
9. according to the additive of claim 7 or 8, which is characterized in that comprising at least one according to claim 1 to one of 5 Polypeptide.
10. additive according to claim 8, which is characterized in that the auxiliary agent is at least one inert carrier.
11. additive according to claim 8, which is characterized in that the auxiliary agent be at least one inert carrier and other at Point.
12. additive according to claim 11, which is characterized in that the other compositions be vitamin and/or minerals and/or Enzyme and/or other components for being used to that mycotoxin to be made to detoxify.
13. additive according to claim 9, which is characterized in that in the additive, with highest 10, the concentration of 000U/g Comprising it is at least one according to claim 1 to one of 5 polypeptide.
14. additive according to claim 9, which is characterized in that in the additive, with highest 1, the concentration packet of 000U/g Containing it is at least one according to claim 1 to one of 5 polypeptide.
15. additive according to claim 9, which is characterized in that in the additive, the concentration with highest 100U/g includes It is at least one according to claim 1 to one of 5 polypeptide.
16. additive according to claim 9, which is characterized in that in the additive, the concentration with highest 10U/g includes It is at least one according to claim 1 to one of 5 polypeptide.
17. according to the additive of claim 7 or 8, which is characterized in that the additive through packing or coated form to deposit In.
18. the polypeptide of amino acid sequence or its functional variant thereof with SEQ ID No.11 is used to raise with hydrolysis method cracking The purposes of zearalenone and/or at least one zearalenone derivative in material, in food or in distiller's dried grain, The zearalenone derivative is selected from α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S and ZAN, wherein the function Sequence identity between property variant and the amino acid sequence is at least 83%.
19. purposes according to claim 18, wherein the feed is the feed for pig, poultry and aquaculture.
20. the method for cracking zearalenone and/or at least one zearalenone derivative with hydrolysis method, institute It states zearalenone derivative and is selected from α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S and ZAN, which is characterized in that institute State zearalenone and/or at least one zearalenone derivative by with SEQ ID No.11 amino acid sequence or The polypeptide of its functional variant thereof hydrolyzes, wherein the sequence identity between the functional variant thereof and the amino acid sequence is At least 83%.
21. method according to claim 20, which is characterized in that used in the additive according to one of claim 7 to 17 The polypeptide.
22. method according to claim 21, which is characterized in that by the polypeptide or additive with by zearalenone and/ Or mutually blended by the feed or food that at least one zearalenone derivative pollutes, make the contaminated feed or food It is in contact with moisture, and the polypeptide or additive hydrolysis corn included in the contaminated feed or food are red Mould ketenes and/or at least one zearalenone derivative.
23. according to the method for one of claim 20 to 22, which is characterized in that at least 70% zearalenone and/ Or at least one zearalenone derivative is hydrolyzed.
24. according to the method for one of claim 20 to 22, which is characterized in that at least 80% zearalenone and/ Or at least one zearalenone derivative is hydrolyzed.
25. according to the method for one of claim 20 to 22, which is characterized in that at least 90% zearalenone and/ Or at least one zearalenone derivative is hydrolyzed.
Embodiment 1: coding can crack the multicore glycosides of the polypeptide of ZEN and/or at least one ZEN derivative with hydrolysis method Modification, clone and the expression of acid
To specifications, " Quick-change Site-directed Mutagenesis Kits " is used (Stratagene), by means of PCR, amino acid replacement, insertion or missing are carried out by the mutation of nucleotide sequence.Alternatively Complete nucleotide sequence (GeneArt) has also been bought on ground thus.It is being generated by means of PCR mutagenesis or bought from GeneArt Nucleotides sequence be listed on amino acid levels and optionally comprise in addition the end C- or the end N- 6 × His label, and by means of Standard method is integrated into for carrying out table in Escherichia coli (E.coli) or pichia pastoris yeast (P.pastoris) It among the expression vector reached, is transformed into Escherichia coli or pichia pastoris yeast, and finishes in Escherichia coli or Pasteur red (J.M.Cregg, Pichia Protocols, the second edition, ISBN-10:1588294293,2007 are expressed in yeast； J.Sambrook et al., 2012, Molecular Cloning, A Laboratory Manual, the 4th edition, Cold Spring Harbor), wherein or the task consider any other suitable host cell.
Title " expression vector " relates to the DNA construct of expressing gene in vivo or in vitro.Particularly, the title Cover such DNA construct, be suitable for for the nucleotide sequence of coding said polypeptide being transferred in host cell, so as to There is integrated into genome or it is free be present in dye in extrapersonal space, and express coding said polypeptide in the cell Nucleotide sequence and the polypeptide is optionally extracted from cell.
Title " host cell " is related to all such cells, or includes nucleotide sequence or packet to be expressed Containing expression vector, and polypeptide according to the present invention can be prepared.Particularly, which covers prokaryotic cell and/or eukaryon is thin Born of the same parents, preferably pichia pastoris yeast, Escherichia coli, bacillus subtilis, streptomyces (Streptomyces), Hansenula yeast Belong to (Hansenula), trichoderma (Trichoderma), lactobacillus (Lactobacillus), aspergillus (Aspergillus), plant cell and/or the spore of bacillus (Bacillus), trichoderma or aspergillus.
In order to measure the catalysis characteristics of polypeptide, soluble cell lysate, Huo Zhe are considered in the case where Escherichia coli Culture supernatants are considered in the case where pichia pastoris yeast.In order to measure KMValue, vmax、kcatAnd specific activity, by means of mark Quasi- method is selectively enriched with the polypeptide by nickel-Sepharose column in a manner of chromatography.The measurement of protein concentration by In standard method to carry out or using BCA method (Pierce BCA Protein Assay KitProd#23225), however Preferably using the photometry implemented with the specific extinction coefficient about respective protein, the specific extinction coefficient is used in Online available program " ProtParam " calculates (Gasteiger E. on http://web.expasy.org/protparam Et al., Protein Identification and Analysis Tools on the ExPASy Server, John M.Walker (editor): The Proteomics Protocols Handbook, Humana Press, 2005,571-607 Page).