WO2017097132A1 - 定点突变改造的酵母二肽基肽酶iii - Google Patents

定点突变改造的酵母二肽基肽酶iii Download PDF

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WO2017097132A1
WO2017097132A1 PCT/CN2016/107454 CN2016107454W WO2017097132A1 WO 2017097132 A1 WO2017097132 A1 WO 2017097132A1 CN 2016107454 W CN2016107454 W CN 2016107454W WO 2017097132 A1 WO2017097132 A1 WO 2017097132A1
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dipeptidyl peptidase
site
peptidase iii
amino acid
yeast dipeptidyl
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PCT/CN2016/107454
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刘大岭
姚冬生
吴曦阳
谢春芳
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暨南大学
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C12N1/185—Saccharomyces isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/14—Dipeptidyl-peptidases and tripeptidyl-peptidases (3.4.14)
    • C12Y304/14004—Dipeptidyl-peptidase III (3.4.14.4)

Definitions

  • the present invention relates to a dipeptidyl peptidase III, and more particularly to a site-directed mutagenized yeast-derived dipeptidyl peptidase III.
  • Dipeptidase III (DPP S III, EC 3.4.14.4), such as dipeptidase III from yeast, dipeptidase III from humans, dipeptidase III from rat, dipeptidase III from rabbit, etc. It is a group of metalloproteinases containing a special HEXXGH zinc finger structure in the molecule, and has a peptidase which hydrolyzes the amino terminus of the polypeptide chain to cleave the dipeptide. DPPIII is physiologically related to the metabolism of important physiologically active peptides such as enkephalin and angiotensin II, angiotensin III, and melanin. DPP S is present in various mammalian tissues.
  • Dipeptidyl peptidases are classified into different types of DPP I to DPP IV depending on the localization of the subcellular and the sensitivity of the specific non-nuclear inhibitor.
  • DPP III can selectively hydrolyze dipeptide residues from the N-terminus of the polypeptide chain or protein, such as: Arg-Arg-, Ala-Arg-, or Tyr-Gry.
  • Yeast-derived DPP III consists of 712 amino acids, which are catalytic metal ions. Its amino acid sequence has a homology of 37% with Aflatoxin monooxygenase (AFMO), but does not have the function of oxidative decomposition of 6-methoxybisfuranocoumarin.
  • AFMO Aflatoxin monooxygenase
  • Aflatoxin is mainly a highly toxic secondary metabolite produced by fungi such as Aspergillus flavus and Aspergillus parasiticus.
  • the identified aflatoxin molecules have eight major structures, among which the most toxic aflatoxin B1 (also known as 6) -Methoxydifuranocoumarin) is considered to be a potential class of strong carcinogenic mutagens that are extremely harmful to humans.
  • a large intake can cause acute poisoning and even death in humans and animals; long-term intake in small doses It is teratogenic, mutagenic and carcinogenic, even if it is extremely toxic at dozens of ppb; Group I aflatoxins contain a furan double bond structure.
  • Aflatoxin monooxygenase is an enzyme that oxidatively decomposes 6-methoxybisfuranocoumarin. It has been found that the process of oxidative decomposition of 6-methoxybisfuranocoumarin by aflatoxin monooxygenase is: electrons are transferred from the substrate molecules to oxygen, water is reduced to hydrogen peroxide, and the substrate is oxidized. Further, the furan double bond in the molecule is opened. In this process, the electrons are transferred by the valence state of the valence ions in the molecule.
  • the divalent metal ions bound to the AFMO capture an electron of the substrate, become a monovalent ion itself, and then become unstable.
  • the electron of the monovalent ion will be captured
  • oxygen it itself transforms into a stable divalent ion, and the oxygen molecule acquires an electron to generate hydrogen peroxide with the participation of water molecules, while the substrate is converted into its epoxide.
  • the epoxide undergoes an oxidative hydrolysis reaction with the action of hydrogen peroxide, eventually breaking the furan double bond in the substrate molecule.
  • Aflatoxin monooxygenase is a biological enzyme currently reported for the detoxification of aflatoxin. Therefore, the discovery and manufacture of new enzymes with oxidative decomposition of 6-methoxybisfuranocoumarin are of great practical significance for the development of aflatoxin reduction technology.
  • a primary object of the present invention is to provide a site-directed mutant yeast dipeptidyl peptidase III which has an oxidative decomposition function for 6-methoxybisfuranocoumarin.
  • a site-directed mutagenized yeast dipeptidyl peptidase III of the present invention is a yeast dipeptidyl peptidase III derived from the yeast Saccharomyces cerevisiae S288c having the amino acid sequence of SEQ ID NO. 1 (NCBI database accession number NM_001183312) a yeast dipeptidyl peptidase III mutant produced by the substitution of a plurality of amino acid substitutions for oxidative decomposition of 6-methoxybisfuranocoumarin, said amino acid substitutions including positions 570, 572 and 574 Replacement.
  • the amino acid at position 570 is substituted with alanine (Alanine, ALA, A) for lysine (Lysine, Lys, K).
  • the amino acid substitution at position 572 is to replace glycine (Glycine, Gly, G) with lysine (Lysine, Lys, K);
  • the amino acid substitution at position 574 is to replace the color ammonia with histidine (Histidine, His, H).
  • Acid (Tryptophan, Trp, W) the amino acid sequence of the site-directed mutagenized yeast dipeptidyl peptidase III mutant is SEQ ID NO.
  • myDPP site-directed mutagenized yeast dipeptidyl peptidase III
  • myDPP site-directed mutagenized yeast dipeptidyl peptidase III
  • the present invention provides a DNA molecule encoding the site-directed mutagen-modified yeast dipeptidyl peptidase III of the present invention.
  • nucleotide sequence of the DNA molecule of the present invention is SEQ ID NO.
  • Another object of the present invention is to provide a vector comprising the DNA molecule of the present invention.
  • Both the above vectors and host cells can be prepared by techniques well known in the art.
  • the present invention also provides a method for producing a site-directed mutagen-modified yeast dipeptidyl peptidase III according to the present invention, comprising: cultivating the host cell of the present invention under conditions suitable for expression of dipeptidyl peptidase III, and The yeast dipeptidyl peptidase III is isolated from the culture medium.
  • the DNA molecule of the present invention When the DNA molecule of the present invention is inserted into the vector in a suitable orientation and the correct reading frame or into the host cell, the DNA molecule can be expressed in any eukaryotic or prokaryotic expression system.
  • Many host-vector systems can be used to express protein coding sequences.
  • Host-vector systems include, but are not limited to, bacteria transformed with phage, plasmid or cosmid; microorganisms containing yeast vectors, such as yeast; mammalian cell systems infected with viruses; insect cell systems infected with viruses; infected with bacteria Plant cell system.
  • Preferred vectors of the invention include viral vectors, plasmids, cosmids or oligonucleotides.
  • a preferred host of the invention is a eukaryotic system such as Pichia pastoris; a preferred protein expression method of the invention is Pichia pastoris methanol-induced secretion expression.
  • the present inventors succeeded in obtaining a site-directed mutagenized yeast dipeptidyl peptidase III, which was confirmed by activity identification experiments that the dipeptidyl peptidase III mutant has an oxidation which is not possessed by wild-type dipeptidyl peptidase III. Decomposes the biological activity of 6-methoxybisfuranocoumarin, and the biological activity reaches the level of application for feed and its additive processing, food and its additive processing, and drug development.
  • Another object of the present invention is to provide an application of the site-directed mutagenized yeast dipeptidyl peptidase III for the elimination of 6-methoxybisfuranocoumarin in the preparation of feed and its additives or foods and additives thereof .
  • the site-directed mutagenized yeast dipeptidyl peptidase III of the present invention can be added as a detoxifying agent to the feed for detoxification of the feed, or an immobilized enzyme can be used for Detoxification of foods such as peanut oil, or preparation of probiotics or probiotic microcapsules capable of expressing the enzyme for detoxification of food, grain, oil, feed, and the like.
  • Another object of the present invention is to provide the use of the yeast dipeptidyl peptidase III engineered by the site-directed mutagenesis for the preparation of a medicament for preventing a disease induced by 6-methoxybisfuranocoumarin.
  • the yeast dipeptidyl peptidase III engineered by the site-directed mutagenesis described in the present invention can be used to obtain a drug for preventing a disease induced by 6-methoxybisfuranocoumarin (for example, a tumor). .
  • Figure 1 is a diagram showing the identification of the recombinant plasmid myDPP expression plasmid.
  • Figure 2 shows the results of purification of recombinant myDPP and recombinant wtyDPP.
  • wtyDPP means wild type yeast dipeptidyl peptidase III, the gene of which is represented by italic wtyDPP.
  • myDPP denotes the mutant yeast dipeptidyl peptidase III, the gene of which is represented by italic myDPP.
  • the present invention uses Saccharomyces cerevisiae S288c dipeptidyl peptidase gene (NCBI database NM_001183312) sequence as a reference, adding 5'-GTC GAATTC -3' at the 5' end and adding 3'- CCTAGG GAC-5' at the 3' end, ( GAATTC ) is the restriction enzyme site EcoRI, ( GGATCC ) is the restriction enzyme site BamHI.
  • the wtyDPP gene was synthesized by artificial total synthesis.
  • the present invention uses the dipeptidyl peptidase gene (NM_001183312) sequence of Saccharomyces cerevisiae S288c as a reference, and the amino acid at position 570 is substituted with alanine (Alanine, ALA, A), and the amino acid at position 572 is treated with lysine (Lysine). , Lys, K) substitution, the amino acid at position 574 is substituted with histidine (Histidine, His, H), and 5'-GTC GAATTC -3' is added at the 5' end. 3'- CCTAGG GAC-5 is added at the 3' end. ', ( GAATTC ) is the restriction enzyme site EcoRI, and ( GGATCC ) is the restriction enzyme site BamHI.
  • the myDPP target gene was synthesized by artificial total synthesis.
  • alanine (Alanine, ALA, A) at position 570, lysine (Lysine, Lys, K) at position 572, and histidine at position 574 (Histidine, His, H) With 576th glutamine (Glutamine, Gln, Q), 578th histidine (Histidine, His, H), 579th methionine (Methionine, Met, M), 580th Glutamine (Glnamine, Gln, Q), alanine (Alanine, ALA, A) at position 581, and arginine (Arginine, Arg, R) at position 582 form an AXKXHXQXHMQAR (where A is C) Amino acid, R is arginine, X is any amino acid) sequence.
  • Example 1 Gene cloning was carried out in a conventional manner (Sambrook, et al. 2001, Molecular Cloning A Laboratory Manual. Cold Spring Harbor Labroratory Press. USA), and the obtained Example 1 was obtained.
  • the wtyDPP and myDPP were cloned into pHIL-S1 to construct the expression plasmid pHIL-S1-wtyDPP and the expression plasmid pHIL-S1-myDPP.
  • the cloned target gene was identified by enzyme digestion and sequencing.
  • the recombinant plasmid pHIL-S1 containing myDPP was constructed by EcoRI+BamHI double-digestion plasmid pHIL-S1 and the target fragment myDPP.
  • the digested product was subjected to 0.8% agarose gel electrophoresis and recovered by gelation.
  • the ligation of plasmid pHIL-S1 and myDPP was carried out using T4 DNA ligase.
  • E.coli DH5 ⁇ competent cells were prepared by CaCl 2 method, transformed into DH5 ⁇ competent cells, screened for transformants, and extracted with alkali.
  • the recombinant plasmid pHIL-S1-myDPP was identified by digestion with EcoRI+BamHI, HindIII and SacI.
  • Plasmid DNA was extracted by picking up a recombinant plasmid PEG purification method (Sambrook, et al. 2001, Molecular Cloning A Laboratory Manual. Cold Spring Harbor Labroratory Press. USA). T7 and SP6 were used as sequencing primers, and the DNA automatic sequencer was used to measure in the forward and reverse directions. The results of enzyme digestion are shown in Figure 1.
  • the restriction vector of the recombinant vector pHIL-S1-myDPP was identified. BamHI and EcoRI double digestion (sample 1) cut a band at about 2100 bp, HindIII single digestion (sample 2), SacI single enzyme. Cut (sample 3).
  • the recombinant plasmid pHIL-S1 containing wtyDPP was constructed by replacing the target fragment myDPP in the construction of myDPP-containing recombinant plasmid pHIL-S1 with wtyDPP, and the other procedures were the same as those of the recombinant plasmid pHIL-S1 containing myDPP. .
  • Example 3 Expression of recombinant myDPP and recombinant wtyDPP
  • Recombinant myDPP expression Recombinant plasmid pHIL-S1-myDPP and plasmid pHIL-S1 were digested with SacI, and the digested product was subjected to 0.8% agarose gel electrophoresis, and the linear recombinant plasmid pHIL-S1-myDPP and the enzyme were digested. Plasmid pHIL-S1. Pichia pastoris GS115 was transformed according to the protoplast method in the Pichia Expression Kit (Invitrogen Inc., USA) manual, and Mut + transformants were selected.
  • the culture medium was analyzed by SDS-PAGE.
  • the expression of the target protein band was observed in the supernatant of the culture medium after induction transformation.
  • the control strain which transformed the empty plasmid containing no gene of interest was induced under the same conditions for 96 hours, and no target protein band was detected in the supernatant. The results are shown in Fig.
  • sample 1 is myDPP, sample 2 is wtyDPP
  • the transformant is induced by methanol, the supernatant of the culture medium shows a distinct protein band; and the negative control strain which transforms the empty plasmid containing no gene of interest Under the same conditions, no target protein was induced in the supernatant.
  • the recombinant plasmid pHIL-S1-myDPP and plasmid pHIL-S1 were digested with SacI, and the plasmid pHIL-S1 was linearized as a control for the following experiment.
  • pHIL-S1 120 total system: 12 ⁇ l Buffer L+8 ⁇ l SacI+100 ⁇ l pHIL-S1 0.8% agarose gel electrophoresis, the gel-removed linear recombinant plasmid pHIL-S1-myDPP and plasmid pHIL-S1.
  • the cultured cells were resuspended in 200 ml of sterile water and transferred to two sterile 10 ml centrifuge tubes;
  • the pellet was resuspended in 150 ⁇ l of SOS and incubated at room temperature for 20 min;
  • the total protein amount in the supernatant cultured for 96 hours was 0.23 mg/ml.
  • the molecular weight of the target protein is consistent with the theoretical value of 78 kDa.
  • Recombinant wtyDPP expression The recombinant plasmid in the expression of recombinant myDPP was replaced with pHIL-S1-wtyDPP, and the other processes were identical to those of recombinant myDPP.
  • the induced fermentation broth was precipitated with 70% saturated (NH 4 ) 2 SO 4 and a precipitate was taken to obtain a crude enzyme sample.
  • the crude enzyme sample was dissolved in an equal volume of PBS. After centrifugation, the supernatant was subjected to a hydrophobic chromatography Phenyl sepharose column, and eluted with a continuous gradient elution buffer to collect the target peak. The solution was desalted, and the PBS solution was equilibrated and concentrated. details as follows:
  • the recombinant expression fermentation broth was added to (NH 4 ) 2 SO 4 powder to 40% saturation, centrifuged at 10000 g for 20 minutes at 4 ° C, and the supernatant was further added with (NH 4 ) 2 SO 4 powder to 70% saturation. Centrifuge at 10,000 g for 20 minutes at 4 °C. That is, a crude enzyme sample is obtained.
  • the solution was desalted and equilibrated with liquid F (0.02 M PBS + 0.5 M NaCl, pH: 7.5) and concentrated to a protein concentration of about 1 mg/ml.
  • the peak of interest was identified by SDS-PAGE electrophoresis.
  • Example 5 Detection of oxidative decomposition of 6-methoxybisfuranocoumarin by myDPP and wtyDPP recombinant protein
  • enzyme activity unit The amount of enzyme required to produce 1 ⁇ mol of H 2 O 2 by one-minute enzyme-catalyzed substrate at 25 ° C.
  • HRP horseradish peroxidase
  • TMB 3,3'-5,5'-tetramethylbenzidine
  • the enzyme activity assay showed that the wtyDPP protein had no decomposition effect on 6-methoxybisfuranocoumarin; The myDPP protein has a decomposing effect on 6-methoxybisfuranocoumarin, and the relative enzyme activity is 33.61 U/mg.

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Abstract

提供定点突变改造的酵母二肽基肽酶III。所述的定点突变改造的酵母二肽基肽酶III由氨基酸序列为SEQ ID NO.1的来源于Saccharomyces cerevisiae S288c的酵母二肽基肽酶III中多个氨基酸取代而产生的突变体，所述的氨基酸取代包括第570、572、574位的取代，以致所述定点突变改造的酵母二肽基肽酶III为对6-甲氧基双呋喃香豆素具有氧化分解作用的酶。所述的定点突变改造的酵母二肽基肽酶III可应用于制备饲料及其添加剂或食品及其添加剂中消除6-甲氧基双呋喃香豆素的产品，还可应用于制备预防由6-甲氧基双呋喃香豆素诱发的疾病的药物。

Description

[根据细则37.2由ISA制定的发明名称]　定点突变改造的酵母二肽基肽酶III 技术领域
本发明涉及一种二肽基肽酶III，特别涉及一种定点突变改造的酵母来源的二肽基肽酶III。
背景技术
二肽酶III(DPPS III，EC 3.4.14.4)，如来自酵母的二肽酶III、来自人类的二肽酶III、来自大鼠的二肽酶III、来自兔的二肽酶III等，是一组分子内含有特殊的HEXXGH锌指结构的金属蛋白酶，具有水解多肽链的氨基末端切掉二肽的肽酶。DPPIII在生理功能上与脑啡肽和血管紧张素II、血管紧张素III、促黑色素等重要的生理活性肽的新陈代谢有关。DPPS存在于各种哺乳动物的组织中，根据亚细胞的定位、特异性地无核抑制剂的敏感性，二肽基肽酶分成不同的类型DPP I～DPP IV。DPP III可以选择性地从多肽链或蛋白质的N-末端水解掉二肽残基，如：Arg-Arg-，Ala-Arg-，或者Tyr-Gry。酵母来源的DPP III由712个氨基酸组成，锌离子是其催化金属离子。其氨基酸序列与黄曲霉毒素单加氧酶(Aflatoxin monooxygenas，AFMO)的同源性达到37％，但不具有对6-甲氧基双呋喃香豆素氧化分解的功能。
黄曲霉毒素主要是由黄曲霉和寄生曲霉等真菌产生的剧毒性次生代谢产物，已经确定的黄曲霉毒素分子的结构主要有八种，其中毒性最强的黄曲霉毒素B1(也称6-甲氧基双呋喃香豆素)被认为是潜在的对人类危害极为突出的一类强致癌诱变剂，一次大量摄入会引起人及动物的急性中毒、甚至死亡；小剂量长期摄入会致畸、致突变和致癌，即使几十ppb的含量仍有极大的毒性；I族黄曲霉毒素含有呋喃双键结构。目前，已经发现的对6-甲氧基双呋喃香豆素具有分解作用的生物酶极少。黄曲霉毒素单加氧酶(AFMO)是一个对6-甲氧基双呋喃香豆素有氧化分解作用的酶。经研究发现黄曲霉毒素单加氧酶氧化分解6-甲氧基双呋喃香豆素的过程为：从底物分子中获得电子传递给氧，使水还原成过氧化氢，将底物氧化并进一步使分子中的呋喃双键开环。在这个过程中，由分子内的变价离子的价态变化来实现电子的传递，首先，结合在AFMO上的二价金属离子夺得底物的一个电子，自身变成一价离子，然后不稳定的一价离子将夺得的这个电子传 递给氧，自身又转变成稳定的二价离子，氧气分子获得一个电子在水分子的参与下生成过氧化氢，同时底物转变为它的环氧化物。然后，该环氧化物伴随着过氧化氢的作用发生氧化水解反应，最终使底物分子中的呋喃双键断开。黄曲霉毒素单加氧酶是目前已报道的用于黄曲霉毒素解毒的生物酶。因此，发现和制造新的对6-甲氧基双呋喃香豆素具有氧化分解作用的酶，对发展黄曲霉毒素消减技术具有十分重要的现实意义。
发明内容
本发明的首要目的是提供一种定点突变的酵母二肽基肽酶III，该酵母二肽基肽酶III突变体对6-甲氧基双呋喃香豆素具有氧化分解功能。
本发明所述的一种定点突变改造的酵母二肽基肽酶III是由氨基酸序列为SEQ ID NO.1的来源于酵母Saccharomyces cerevisiae S288c的酵母二肽基肽酶III(NCBI数据库登录号为NM_001183312)中制造多个氨基酸取代而产生的、对6-甲氧基双呋喃香豆素有氧化分解作用的酵母二肽基肽酶III突变体，所述的氨基酸取代包括第570、572和574位的取代。
根据本发明所述的定点突变改造的酵母二肽基肽酶III的进一步特征，所述第570位的氨基酸用丙氨酸(Alanine，ALa,A)取代赖氨酸(Lysine，Lys,K)；第572位的氨基酸取代是用赖氨酸(Lysine，Lys,K)取代甘氨酸(Glycine，Gly，G)；第574位的氨基酸取代是用组氨酸(Histidine,His,H)取代色氨酸(Tryptophan,Trp,W)；所述的定点突变改造的酵母二肽基肽酶III突变体的氨基酸序列为SEQ ID NO.2。
经实验验证，本发明所述的定点突变改造的酵母二肽基肽酶III(以下缩写为“myDPP”)具有对于6-甲氧基双呋喃香豆素的氧化分解功能。该突变体酶在pH 6.0、反应温度25℃处理6-甲氧基双呋喃香豆素(100ppb)50分钟后，其对6-甲氧基双呋喃香豆素的氧化分解效率达到90％。该突变体酶的其它酶学性质与野生酶相似。
进一步地，本发明提供了一种DNA分子，其编码本发明所述的定点突变改造的酵母二肽基肽酶III。
优选地，本发明所述的DNA分子的核苷酸序列为SEQ ID NO.3。
本发明的另一个目的是提供一种载体，其含有本发明所述的DNA分子。
本发明的又一个目的是提供一种宿主细胞，其含有本发明所述的DNA分子，或者含有本发明所述的载体。
上述载体和宿主细胞都可以通过本领域公知的技术手段进行制备。
本发明还提供了本发明所述的定点突变改造的酵母二肽基肽酶III的生产方法，包括：在适于二肽基肽酶III表达的条件下培养本发明所述的宿主细胞，并从培养基中分离所述的酵母二肽基肽酶III。
当本发明所述的DNA分子以合适的取向和正确的阅读框插入到所述的载体或者转入所述的宿主细胞中，所述的DNA分子可以在任何真核或者原核表达系统中表达。许多宿主-载体系统都可以用来表达蛋白质编码序列。宿主-载体系统包括但不限于：用噬菌体、质粒或粘粒转化的细菌；含有酵母载体的微生物，如酵母；用病毒感染的哺乳动物细胞系统；用病毒感染的昆虫细胞系统；用细菌感染的植物细胞系统。本发明优选的载体包括病毒载体、质粒、粘粒或者寡核苷酸。
本发明优选的宿主为真核系统如毕赤酵母；本发明优选的蛋白质表达方法为毕赤酵母甲醇诱导分泌表达。
本发明人成功地获得了一种定点突变改造的酵母二肽基肽酶III，通过活性鉴定实验，证明该二肽基肽酶III突变体具有野生型二肽基肽酶III所不具备的氧化分解6-甲氧基双呋喃香豆素的生物活性，且该生物活性达到了应用于饲料及其添加剂加工、食品及其添加剂加工以及药物开发的程度。
本发明的另一目的是提供将所述的定点突变改造的酵母二肽基肽酶III在制备饲料及其添加剂或食品及其添加剂中消除6-甲氧基双呋喃香豆素的产品的应用。结合目前的饲料和食品的加工工艺，可将本发明所述的定点突变改造的酵母二肽基肽酶III作为脱毒剂添加到饲料中进行饲料脱毒，或者制成固定化酶用于如花生油等食品的脱毒，或制成能够表达该酶的益生菌制剂或益生菌微囊等用于食品、粮油、饲料等的脱毒。
本发明的另一目的是提供将所述的定点突变改造的酵母二肽基肽酶III在制备预防由6-甲氧基双呋喃香豆素诱发的疾病的药物的应用。结合目前的常规药物制备工艺，可用本发明所述的定点突变改造的酵母二肽基肽酶III制得到用于预防由6-甲氧基双呋喃香豆素诱发的疾病(例如肿瘤)的药物。
附图说明
图1为重组质粒myDPP表达质粒的鉴定图。
图2为重组myDPP和重组wtyDPP的纯化结果。
具体实施方式
本文中所采用的术语，除非另有说明，均为本领域技术人员所通常理解的含义。以下提供在本发明中使用的一些特殊术语的定义。
“wtyDPP”表示野生型酵母二肽基肽酶III，其基因以斜体wtyDPP表示。
“myDPP”表示突变体酵母二肽基肽酶III，其基因以斜体myDPP表示。
实施例1：wtyDPP和myDPP的合成
本发明以Saccharomyces cerevisiae S288c的二肽基肽酶基因(NCBI数据库NM_001183312)序列作为参考，在5’端加入5’-GTCGAATTC-3’在3’端加入3’-CCTAGGGAC-5’，(GAATTC)为限制酶切位点EcoRI，(GGATCC)为限制酶切位点BamHI。采用人工全合成的方法合成wtyDPP基因。
本发明以Saccharomyces cerevisiae S288c的二肽基肽酶基因(NM_001183312)序列作为参考，将第570位的氨基酸用丙氨酸(Alanine，ALa,A)取代，第572位的氨基酸用赖氨酸(Lysine，Lys,K)取代，第574位的氨基酸用组氨酸(Histidine,His,H)取代，在5’端加入5’-GTCGAATTC-3’在3’端加入3’-CCTAGGGAC-5’，(GAATTC)为限制酶切位点EcoRI，(GGATCC)为限制酶切位点BamHI。采用人工全合成的方法合成myDPP目的基因。
在定点突变改造后，第570位的丙氨酸(Alanine，ALa,A)、第572位的赖氨酸(Lysine，Lys,K)、第574位的组氨酸(Histidine,His,H)与第576位的谷氨酰胺(Glutamine,Gln,Q)、第578位的组氨酸(Histidine,His,H)、第579位的甲硫氨酸(Methionine,Met,M)、第580位的谷氨酰胺(Glutamine,Gln,Q)、第581位的丙氨酸(Alanine，ALa,A)以及第582位的精氨酸(Arginine，Arg,R)形成一个AXKXHXQXHMQAR(其中，A为丙氨酸，R为精氨酸，X为任意氨基酸)序列。
基因合成由商业性公司(例如上海捷瑞生物有限公司)完成。
实施例2：重组质粒wtyDPP和myDPP表达质粒的构建
基因克隆按常规方法(Sambrook,et al.2001,Molecular Cloning A Laboratory Manual.Cold Spring Harbor Labroratory Press.USA)进行，将实施例1所得的 wtyDPP和myDPP分别克隆到pHIL-S1构建成表达质粒pHIL-S1-wtyDPP和表达质粒pHIL-S1-myDPP，克隆后的目的基因经酶切、测序鉴定。
具体做法：
含myDPP的重组质粒pHIL-S1的构建过程为：EcoRI+BamHI双酶切质粒pHIL-S1和目的片断myDPP，酶切产物进行0.8％琼脂糖凝胶电泳，并切胶回收。利用T4DNA连接酶进行质粒pHIL-S1和myDPP的连接。CaCl2法制备E.coli DH5α感受态细胞，转化DH5α感受态细胞，筛选转化子，碱提质粒。用EcoRI+BamHI、HindⅢ、SacI酶切鉴定重组质粒pHIL-S1-myDPP。挑取重组质粒PEG纯化法(Sambrook，et al.2001，Molecular Cloning A Laboratory Manual.Cold Spring Harbor Labroratory Press.USA)提取质粒DNA。以T7和SP6为测序引物，采用DNA自动序列仪，正反向进行测定。酶切结果如图1示，重组载体pHIL-S1-myDPP的酶切鉴定，BamHI、EcoRI双酶切(样品1)切下一条带位于2100bp左右，HindIII单酶切(样品2)，SacI单酶切(样品3)。
含wtyDPP的重组质粒pHIL-S1的构建过程为：将含myDPP的重组质粒pHIL-S1的构建过程中的目的片断myDPP替换为wtyDPP，其他操作过程与含myDPP的重组质粒pHIL-S1的构建过程相同。
实施例3：重组myDPP和重组wtyDPP的表达
重组myDPP的表达：用SacI酶切重组质粒pHIL-S1-myDPP和质粒pHIL-S1，酶切产物进行0.8％琼脂糖凝胶电泳，切胶回收酶切后的线性重组质粒pHIL-S1-myDPP和质粒pHIL-S1。按Pichia Expression Kit(Invitrogen Inc.，美国)手册中的原生质法转化毕赤酵母菌GS115，筛选Mut+转化子。利用甲醇作为唯一碳源对重组菌进行诱导表达(按Pichia Expression Kit手册操作)中，培养液经SDS-PAGE电泳分析表明，转化子经诱导表达后，培养液上清出现明显的目的蛋白带，而转化不含目的基因的空质粒的对照菌在相同的条件下诱导96小时，在上清中未检测到目的蛋白条带。结果如图2所示(样品1为myDPP，样品2为wtyDPP)，转化子经甲醇诱导表达后，培养液上清出现明显的目的蛋白带；而转化不含目的基因的空质粒的阴性对照菌，在相同的条件下诱导在上清中无目的蛋白出现。
具体做法如下：
一、重组子与毕赤酵母的同源重组
(一)线性化质粒
SacI酶切重组质粒pHIL-S1-myDPP和质粒pHIL-S1，同时线性化质粒pHIL-S1是作为下面实验的对照。
酶切pHIL-S1-myDPP(120总体系)：12μl Buffer L+8μl SacI+100μl pHIL-S1-myDPP
酶切pHIL-S1(120总体系)：12μl Buffer L+8μl SacI+100μl pHIL-S1 0.8％琼脂糖凝胶电泳酶切样品，切胶回收酶切后的线性重组质粒pHIL-S1-myDPP和质粒pHIL-S1。
(二)培养用于原生质化的毕赤酵母菌GS115
1.从平板上挑取一个GS115单克隆接种于10mlYPD中，在150ml锥形瓶中，30℃，250-300rpm振荡培养过夜；
2.从昨日培养的的10mlYPD菌液中分别取5，10，20μl接种于200mlYPD中，在500ml的锥形瓶中，250-300rpm振荡培养过夜；
3.检测3个瓶中菌液的OD600值，取OD600＝0.2-0.3的菌液转入离心管中，室温1500×g离心5min，弃上清，收集的细胞用于原生质化。
(三)毕赤酵母菌GS115的原生质化
1.培养收集的细胞重悬于200ml的无菌水中，转移至两个无菌的10ml离心管中；
2.室温下，1500×g离心5min，弃上清；
3.用10ml新鲜配制的SED洗沉淀，室温下，1500×g离心5min，弃上清；
4.用10ml 1M山梨醇洗沉淀，室温下，1500×g离心5min，弃上清；
5.用10mlSCE重悬沉淀；
6.取一管Zymolyase冻融后轻弹管壁，使溶液混匀；
7.取7.5μl Zymolyase加入管中，30℃温育30min；
8.室温下750×g离心10min，收集菌体，弃上清；
9.用10ml 1M山梨醇洗原生质体，轻轻敲打管壁分散沉淀，室温下750×g离心10min，收集菌体，弃上清；
10.用10ml CaS洗菌体，室温下750×g离心10min，收集菌体，弃上清；
11.将沉淀重悬于0.6ml CaS中，此原生质体在30min内必须使用。
(四)原生质法转化毕赤酵母菌GS115
1、分别取100μl毕赤酵母原生质体加入A、B、C 3个无菌15ml离心管中；
2、A管中不加DNA作为阴性对照，B管中加入30μl线性化的原质粒pHIL-S1,C管中加入30μl线性化的重组质粒pHIL-S1-myDPP，室温下温育10min,期间准备一支3ml新鲜配制的PEG/CaT；
3、各管中加入1ml新鲜配制的PEG/CaT，轻轻混匀，室温下温育10min；
4、室温下750×g离心10分钟，弃上清，控干；
5、将沉淀重悬于150μl SOS中，室温温育20min；
6、各管加入850μl 1M山梨醇，准备铺板；
7、涂布RD固体培养基，每板涂布200μl,28-30℃温育倒置培养，转化子约在4-6天出现。
(五)筛选Mut+转化子
1.用无菌牙签挑取His+转化子，分别在MM和MD板上一一对应点菌，同时点上GS115/His+MutsAlbumin和GS115/His+Mut+β-gal作为对照。
2. 28-30℃温育倒置培养2天；
3.两天后，观察对照MM和MD板，若在两板上均生长良好为Mut+，若在MD上生长良好，在MM板上生长少或不生长则为Muts。
(六)重组菌的诱导表达
1.挑取一个His+Mut+转化子的单克隆，接种于25mlBMG，在250ml的锥形瓶中，28-30℃，250-300rpm振荡培养，直至OD600＝2-6(约16-18h)；
2. 1500-3000×g离心5min收集细胞，弃上清，重悬细胞于BMM中至OD600为1.0(约需100-200mlBMM)，在1升的锥形瓶中，28-30℃，250-300rpm继续振荡培养。
3.每24h加100％甲醇，始终浓度至0.5％，保持诱导表达；
4.诱导表达96h的时间。培养液离心2-3min，留取上清放入-80℃保存以用于纯化表达产物。
经诱导96小时培养上清中总蛋白量达0.23mg/ml。目的蛋白的分子量与理论值78kDa相符。
重组wtyDPP的表达：将重组myDPP的表达过程中的重组质粒替换为pHIL-S1-wtyDPP，其他操作过程与重组myDPP的表达流程相同。
实施例4：重组myDPP和wtyDPP的纯化
诱导表达的发酵液经70％饱和(NH4)2SO4沉淀，收取沉淀获得粗酶样品。粗酶样品以等体积的PBS溶解，离心后取上清上疏水色谱层析Phenyl sepharose柱，以连续梯度的洗脱缓冲液洗脱，收集目的峰。透析脱盐，PBS溶液平衡后浓缩。具体如下：
1、硫酸氨沉淀收集粗酶
重组表达发酵液加入(NH4)2SO4粉末至40％饱和度，4℃，10000g离心20分钟，上清继续再加入(NH4)2SO4粉末至70％饱和度。4℃，10000g离心20分钟。即获得粗酶样品。
2、疏水层析：粗酶样品以等体积的0.02M PBS(pH：6.0)液溶解，4000g 4℃离心10分钟，取上清，上Phenyl sepharose柱[Phenyl sepharose 6Fast flow(high sub)，Pharmacia Biotech，Inc]，溶液液(0.02M PBS+30％饱和硫酸铵，pH：6.0)洗至基线，然后以连续梯度的洗脱缓冲液[A液(0.02M PBS+10％饱和硫酸铵，pH：6.0)+B液(0.02M PBS，pH：6.0)]洗脱，收集目的峰。透析脱盐，并以F液(0.02M PBS+0.5M NaCl，pH：7.5)平衡，浓缩至蛋白浓度约为1mg/ml。目的峰用SDS-PAGE电泳鉴定。
实施例5：myDPP及wtyDPP重组蛋白的对6-甲氧基双呋喃香豆素氧化分解作用的检测
酶活力单位的定义：25℃条件下，一分钟酶催化底物产生1μmol的H2O2所需要的酶量。
酶活力测定方法：
在10ml浓度为10μg/ml的酶中加入30μl浓度为100μg/ml的底物(6-甲氧基双呋喃香豆素)，在25℃、pH6.5条件下反应10分钟，然后在反应液中加入200μl浓度为0.34mg/ml的辣根过氧化物酶(HRP)和200μl浓度为5mM的3,3’-5,5’-四甲基联苯胺(TMB)，显色30min，然后在紫外650nm处测量吸光值，计算酶活力单位。
酶活测定结果显示，wtyDPP蛋白对6-甲氧基双呋喃香豆素没有分解作用； myDPP蛋白对6-甲氧基双呋喃香豆素具有分解作用，相对酶活为33.61U/mg。
表1：样品处理方法
Figure PCTCN2016107454-appb-000001
Figure PCTCN2016107454-appb-000002
Figure PCTCN2016107454-appb-000003
Figure PCTCN2016107454-appb-000004
Figure PCTCN2016107454-appb-000005
Figure PCTCN2016107454-appb-000006
Figure PCTCN2016107454-appb-000007
Figure PCTCN2016107454-appb-000008
Figure PCTCN2016107454-appb-000009

Claims (9)

  1. 一种定点突变改造的酵母二肽基肽酶III，其特征在于：它是由氨基酸序列为SEQ ID NO.1的来源于Saccharomyces cerevisiae S288c的酵母二肽基肽酶III中制造多个氨基酸取代而产生的突变体，所述的氨基酸取代包括第570、572、574位的取代，以致所述定点突变改造的酵母二肽基肽酶III为对6-甲氧基双呋喃香豆素具有氧化分解作用的酶。
  2. 根据权利要求1所述的定点突变改造的酵母二肽基肽酶III，其特征在于：所述第570位的氨基酸取代是用丙氨酸(Alanine，ALa,A)取代赖氨酸(Lysine，Lys,K)；第572位的氨基酸取代是用赖氨酸(Lysine，Lys,K)取代甘氨酸(Glycine，Gly，G)；第574位的氨基酸取代是用组氨酸(Histidine,His,H)取代色氨酸(Tryptophan,Trp,W)；所述的定点突变改造的二肽基肽酶的氨基酸序列为SEQ ID NO.2。
  3. 一种DNA分子，其特征在于：其编码权利要求2所述的定点突变改造的酵母二肽基肽酶III。
  4. 根据权利要求3所述的DNA分子，其特征在于：其核苷酸序列为SEQ ID NO.3。
  5. 一种载体，其特征在于：其含有权利要求3或4所述的DNA分子。
  6. 一种宿主细胞，其特征在于：其含有权利要求3或4所述的DNA分子，或者含有权利要求5所述的载体。
  7. 一种根据权利要求1所述的定点突变改造的酵母二肽基肽酶III的生产方法，其特征在于，所述方法包括：在适于二肽基肽酶III表达的条件下培养权利要求6.所述的宿主细胞，并从培养基中分离所述的酵母二肽基肽酶III。
  8. 如权利要求1所述的定点突变改造的酵母二肽基肽酶III在制备饲料及其添加剂或食品及其添加剂中消除6-甲氧基双呋喃香豆素的产品的应用。
  9. 如权利要求1所述的定点突变改造的酵母二肽基肽酶III在制备预防由6-甲氧基双呋喃香豆素诱发的疾病的药物的应用。
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