CN111202940B - Application of zearalenone lactone hydrolase RmZHD in degradation of macrolide antibiotics - Google Patents

Application of zearalenone lactone hydrolase RmZHD in degradation of macrolide antibiotics Download PDF

Info

Publication number
CN111202940B
CN111202940B CN201911327678.XA CN201911327678A CN111202940B CN 111202940 B CN111202940 B CN 111202940B CN 201911327678 A CN201911327678 A CN 201911327678A CN 111202940 B CN111202940 B CN 111202940B
Authority
CN
China
Prior art keywords
rmzhd
gly
leu
zearalenone
rhf
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201911327678.XA
Other languages
Chinese (zh)
Other versions
CN111202940A (en
Inventor
文继开
孙天钰
邓诣群
吴骏
陈庆梅
母培强
邓凤如
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
South China Agricultural University
Original Assignee
South China Agricultural University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by South China Agricultural University filed Critical South China Agricultural University
Priority to CN201911327678.XA priority Critical patent/CN111202940B/en
Publication of CN111202940A publication Critical patent/CN111202940A/en
Application granted granted Critical
Publication of CN111202940B publication Critical patent/CN111202940B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
    • A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
    • A62D2101/20—Organic substances
    • A62D2101/28—Organic substances containing oxygen, sulfur, selenium or tellurium, i.e. chalcogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Plant Pathology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Emergency Management (AREA)
  • Business, Economics & Management (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses application of zearalenone lactone hydrolase RmZHD in degradation of macrolide antibiotics. The research of the invention finds that zearalenone lactone hydrolase RmZHD can efficiently degrade macrolide antibiotics and can be used for reducing antibiotic pollution in the environment. Meanwhile, the invention also provides a recombinase RmZHD-rHF containing zearalenone lactone hydrolase RmZHD, which can further improve the activity and expression of the enzyme RmZHD and has a larger application prospect.

Description

Application of zearalenone lactone hydrolase RmZHD in degradation of macrolide antibiotics
Technical Field
The invention relates to the technical field of biology, and in particular relates to application of zearalenone lactone hydrolase RmZHD in degradation of macrolide antibiotics.
Background
Antibiotics are widely used in the fields of disease prevention and treatment in animal husbandry and animal and plant disease and pest control, and the like. The antibiotics can be left in the animal body due to improper use method or illegal overdose addition. Only a small part of the antibiotics are metabolized and absorbed after entering the animal body or the human body, but more than half of the antibiotics are biologically digested and absorbed and then are discharged to the outside of the body in the form of parent bodies or metabolites through urine and excrement of the animals.
The macrolide antibiotics are the classical antibiotics commonly used in clinic, have macrolide structures in molecules and are mostly basic lipophilic compounds. Among them, tylosin is mainly used for clinically treating and preventing various respiratory tract, intestinal tract, reproductive tract and motion system infections caused by pathogens such as mycoplasma, staphylococcus aureus, pyogenes, pneumococcus, erysipelas bacillus, haemophilus parasuis, neisseria meningitidis, pasteurella, spirochete, coccidiosis and the like. Such as: chronic respiratory disease of poultry, infectious rhinitis of chicken, avian cystitis, infectious sinusitis, salpingitis, mycoplasma pneumonia of swine, atrophic rhinitis, swine dysentery with red feces of swine, gastroenteritis, swine erysipelas, mycoplasma arthritis, intractable diarrhea of livestock and poultry, necrotic enteritis, endometritis, suppurative infection of livestock external genitalia, caprine pleuropneumonia, abortion of ewe, beef cattle liver abscess, and cattle and sheep foot rot. Also can be used for purifying mycoplasma in breeding poultry farms such as egg injection and egg dipping. And has good curative effect on preventing and treating secondary infection of mycoplasma of livestock and poultry in outbreak of toxic diseases, and is a first choice medicament for treating and preventing mycoplasma infection of livestock and poultry in the world. The erythromycin has wide antibacterial spectrum, and is effective on most gram-positive bacteria, part gram-negative bacteria and some atypical pathogenic bacteria. Erythromycin has strong antibacterial activity against Staphylococcus (including enzyme-producing strain), various groups of Streptococcus, Diplococcus pneumoniae, Bacillus anthracis, Clostridium tetani, Diptheria, gonococcus, meningococcus, Bacillus influenzae, Bordetella, Campylobacter jejuni, Legionella, Listeria, and Actinomyces israelensis; for treponema pallidum, mycoplasma pneumoniae, leptospira and rickettsia; chlamydia and the like also have better inhibiting effect. The degradation of the microorganism is an important way for decomposing and transforming the medicine in the food, and the utilization of the microorganism to treat the medicine pollution is an effective method, which has shown good application prospect. At present, there are reports on enzymes or related substances which can degrade macrolide antibiotics with high efficiency.
Zearalenone degrading enzyme is a known zearalenone lactone hydrolase, and can open lactone rings in zearalenone molecules, so that the open zearalenone can not be combined with estrogen receptors any more, and the effect of toxin degradation is achieved. The picroprione et al (2018) disclose a zearalenone hydrolase RmZHD from Rhinocladiella mackenziei (the expression and purification of zearalenone hydrolase from Rhinocladiella mackenziei and its enzymological properties [ J ] microbiological report 2018,45(12):51-57 ]); however, the application of the zearalenone hydrolase RmZHD in degradation of macrolide antibiotics has not been reported yet.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide application of zearalenone degrading enzyme RmZHD in degrading macrolide antibiotics or preparing products for degrading macrolide antibiotics.
The second purpose of the invention is to provide the application of the zearalenone degrading enzyme RmZHD coding gene in preparing products for degrading macrolide antibiotics.
The third purpose of the invention is to provide a recombinase RmZHD-rHF containing RmZHD, so as to further improve the activity and expression level of the RmZHD enzyme.
The fourth object of the invention is a preparation method of the recombinase RmZHD-rHF.
The fifth purpose of the invention is to provide the application of the recombinase RmZHD-rHF.
The above object of the present invention is achieved by the following technical solutions:
according to the invention, the zearalenone degrading enzyme RmZHD is obtained through in vitro recombinant expression, and the finding that the zearalenone degrading enzyme RmZHD can efficiently degrade macrolide antibiotics. The invention thus firstly protects:
SEQ ID NO: 1 in the formula (1), the zearalenone degrading enzyme RmZHD is applied to degrading macrolide antibiotics or preparing products for degrading macrolide antibiotics.
SEQ ID NO: 2, and the zearalenone degrading enzyme RmZHD coding gene is applied to the preparation of products for degrading macrolide antibiotics.
Zearalenone degrading enzyme RmZHD can hydrolyze macrolide structures of macrolide antibiotics, and the purpose of degradation is achieved. The zearalenone degrading enzyme RmZHD can degrade erythromycin, erythromycin ethylsuccinate, erythromycin stearate, erythromycin ethyl carbonate, erythromycin acestearate, erythromycin lactobionate, erythromycin estolate, azithromycin, roxithromycin, clarithromycin, dirithromycin, tylosin, fluoroerythromycin and the like.
Preferably, the macrolide antibiotic is erythromycin and/or tylosin.
Meanwhile, in order to further improve the activity and expression quantity of the RmZHD enzyme, the RmZHD protein is creatively fused to the N end of the human ferritin to obtain a recombinase RmZHD-rHF, and the amino acid sequence of the recombinase RmZHD-rHF is shown as SEQ ID NO: 3, respectively. The recombinase RmZHD-rHF can be self-assembled in vitro to form a cage-shaped nanoparticle structure, and the enzyme activity of the cage-shaped nanoparticle structure is improved by 2-4 times compared with that of the free enzyme RmZHD.
Preferably, the diameter of the cage-shaped nanoparticle structure formed by in vitro self-assembly of the recombinase RmZHD-rHF is 11-13 nm.
The preparation method of the recombinase RmZHD-rHF comprises the following steps of firstly constructing an expression vector pET28a-RmZHD-rHF, then transforming competent cells, inducing IPTG, centrifugally collecting thalli, after resuspending and crushing the thalli, collecting supernatant, purifying and eluting to obtain the recombinase RmZHD-rHF.
Preferably, the sequence of the amplification primer for constructing the expression vector pET28a-RmZHD-rHF is shown as SEQ ID NO: 4 to 7.
Preferably, in the bacterial liquid OD600When the concentration is 0.5 to 0.7 (preferably 0.6), the inducer IPTG is added to the mixture to make the final concentration be 0.1 to 0.3mM (preferably 0.2mM), 16 to 18 ℃ (preferably 16 ℃), and 100 to 200rpm (preferably 150rpm) for 24 to 48 hours (preferably 36 hours).
Compared with a free enzyme RmZHD, the histone deacetylase RmZHD-rHF has better macrolide antibiotic degradation activity. Therefore, the invention also claims the application of the recombinase RmZHD-rHF in degrading macrolide antibiotics or preparing products for degrading macrolide antibiotics.
Preferably, the macrolide antibiotic is erythromycin and/or tylosin.
A product for degrading a macrolide antibiotic, said product comprising the recombinase RmZHD-rHF.
Preferably, the product is a medicament or a kit.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides application of zearalenone lactone hydrolase RmZHD in degradation of macrolide antibiotics. The research of the invention finds that zearalenone lactone hydrolase RmZHD can efficiently degrade macrolide antibiotics and can be used for reducing antibiotic pollution in the environment. Meanwhile, the invention also provides a recombinase RmZHD-rHF containing zearalenone lactone hydrolase RmZHD, which can further improve the activity and expression of the enzyme RmZHD and has a larger application prospect.
Drawings
FIG. 1 shows the SDS-PAGE results of recombinant enzyme RmZHD-rHF and free enzyme RmZHD. M: marker; 1: RmZHD; 2: RmZHD-rHF.
FIG. 2 is a transmission electron micrograph of recombinase RmZHD-rHF.
FIG. 3 is a plot of the growth of Bacillus under erythromycin treatment.
FIG. 4 is a plot of Bacillus growth under tylosin treatment.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 in vitro expression of recombinase RmZHD-rHF and free enzyme RmZHD
1. Expression vector construction
Design strategy for fusion proteins: human ferritin (rHF) is used as a fusion partner, zearalenone degrading enzyme (RmZHD) is placed at the N end of rHF for expression, and RmZHD and rHF are connected through a connecting peptide (linker).
Construction of expression vector: designing specific amplification primers (table 1) according to sequences of RmZHD and pET28a-rHF, respectively introducing Nhe I and BamH I enzyme cutting sites at two ends of RmZHD gene, and carrying out mass amplification on the RmZHD sequence (RmZHD sequence is amplified by using primers 3 and 4 to construct pET28a-RmZHD vector, RmZHD sequence is amplified by using primers 3 and 5 to construct pET28a-RmZHD-rHF vector); the nucleotide sequence of linker (GGGGS) is introduced at the 5' end of rHF gene, and BamH I and Hind III sites are introduced at both ends (rHF sequence is amplified by primers 1 and 2 for constructing pET28a-RmZHD-rHF vector), and PCR conditions and program are referred to tables 2 and 3. And then respectively recovering RmZHD fragments and a pET28a vector by using Nhe I and BamH I double enzyme digestion, connecting enzyme digestion products, transferring the enzyme digestion products into E.coli DH5 alpha, screening positive clones and sequencing, wherein sequencing results show that the vector pET28a-RmZHD is successfully constructed, amplifying and culturing correct positive clones, and storing extracted plasmids for later use. And then double enzyme digestion of pET28a-RmZHD and the amplified rHF fragment is carried out by using BamH I and Hind III, the enzyme digestion products are connected, E.coli DH5 alpha is transferred, positive clone is screened and sequenced, the sequencing result shows that an expression vector pET28a-RmZHD-rHF is successfully constructed, the correct positive clone is subjected to amplification culture, and the extracted plasmid is stored for later use.
TABLE 1 primer sequences
Figure BDA0002328799960000041
Figure BDA0002328799960000051
TABLE 2 PCR conditions
Figure BDA0002328799960000052
TABLE 3 PCR procedure
Figure BDA0002328799960000053
2. Expression purification
(1) The successfully constructed expression vectors pET28a-RmZHD-rHF and pET28a-RmZHD were transferred into E.coli BL21(DE3) competent cells by heat shock method, single colonies were picked up in LB medium containing 50. mu.g/mL kanamycin, after shaking culture at 37 ℃ and 200rpm for 5 hours, transferred at a ratio of 1:100 into 500mL LB medium containing 50. mu.g/mL kanamycin, and shaken at 37 ℃ and 200rpm to OD600When the concentration was about 0.6, the inducer IPTG was added to a final concentration of 0.2mM, and the mixture was induced at 16 ℃ and 150rpm for 36 hours. Centrifuging at 4 deg.C and 5000rpm for 10min, discarding supernatant, and collecting thallus. Resuspending each gram of bacteria with 20mL of binding buffer (25mM Tris, 150mM sodium chloride, pH7.5), carrying out ultrasonication, centrifuging at 4 ℃ and 6000rpm for 15min after crushing, collecting supernatant, filtering the supernatant with a 0.22 mu m filter membrane, purifying by nickel ion affinity chromatography (Ni-NTA), carrying out gradient elution, collecting eluate in different tubes, and carrying out SDS-PAGE electrophoresis. The SDS-PAGE electrophoresis result of the recombinant protein is shown in figure 1, which shows that RmZHD-rHF and RmZHD protein with higher purity are obtained by successful purification.
(2) Transmission Electron Microscopy (TEM) detection of aggregated form of recombinase RmZHD-rHF
Dripping 20 mu L of RmZHD-rHF solution and 20 mu L of negative dyeing liquid on the smooth surface of the sealing film to form a raised small drop; slightly clamping the edge of the copper mesh, and covering the surface of the carbon film on the sample solution to enable the sample to be adsorbed on the surface of the carbon film for 3-5 min; carefully clamping the copper mesh, sucking liquid from the edge of the copper mesh by using filter paper, covering the surface of the negative dyeing liquid with a carbon film surface of the copper mesh after water drops on the surface of the copper mesh cannot be observed by naked eyes, and lasting for 5-7 min. Similarly, after absorbing liquid from the edge by using filter paper, placing the liquid in a plate paved with a layer of filter paper, and marking; and standing for 2-4 h, and performing TEM representation.
The self-assembly of RmZHD-rHF to form a cage-shaped nanoparticle structure is characterized by an electron microscopy technology, and the result is shown in figure 2: the particle regular structure with the diameter of about 11.84 +/-0.56 nm (N is 30) shows that the fusion of RmZHD at the N end of rHF does not influence the self-assembly of human ferritin to form a cage-shaped nano structure, and the separated and purified RmZHD-rHF is successfully self-assembled in vitro to form the cage-shaped nano particle structure.
Example 2 recombinant enzyme RmZHD-rHF and free enzyme RmZHD antibiotic degradation Activity test
The growth curves were tested for the degradation activity of the recombinase RmZHD-rHF and the free enzyme RmZHD on macrolide antibiotics (erythromycin, tylosin for example).
(1) The minimum inhibitory concentration of erythromycin is determined by a drug sensitivity test (MIC).
(2) Bacillus (ATCC 29213) was cultured at a drug concentration of 1/2MIC, and with LB broth as a control, antibiotics after RmZHD treatment, antibiotics after RmZHD-rHF treatment, RmZHD and RmZHD-rHF were added as experimental groups, respectively.
(3) According to the following steps: 100 of the cells were inoculated with bacteria, incubated at 37 ℃ and 200rpm, and OD was measured every 1 hour600And recording the measured value.
(4) The tylosin growth curve was determined in the same manner as for erythromycin.
As a result: the growth curve of the bacillus under the erythromycin treatment is shown in figure 3, the growth curve of the bacillus under the tylosin treatment is shown in figure 4, the growth of the bacillus is inhibited after the erythromycin and the tylosin are added, and the growth inhibition effect of the antibiotic on the bacteria is reduced after the zearalenone degrading enzyme recombinase RmZHD-rHF and the free enzyme RmZHD are added, so that the recombinase RmZHD-rHF and the free enzyme RmZHD have certain degradation capability on the macrolide antibiotic (erythromycin and tylosin).
Example 3 enzyme Activity assay of recombinase RmZHD-rHF and free enzyme RmZHD
Full wavelength scans of erythromycin and tylosin and the enzyme treated product were detected by using an Shimadzu UV-2550 spectrophotometer. After scanning, a specific ultraviolet absorption peak appears at 210nm after the zearalenone degrading enzyme degrades, and the metabolism dynamics of the zearalenone degrading enzyme on erythromycin is determined by determining the increase of a product at 210 nm. The tylosin is scanned, and the specific absorption peak at 290nm is reduced, and the metabolic kinetics of the zearalenone degrading enzyme on the tylosin is determined by measuring the reduction of the substrate at 290 nm.
The standard reaction system contained 50nM RmZHD-rHF or RmZHD and different concentrations of erythromycin (enzyme concentration 10nM when substrate is tylosin), 25mM Tris-HCl and 150mM NaCl in a total volume of 800. mu.L. The absorption value is zeroed before the enzyme is added, the reaction is started after the enzyme is added and degraded and mixed evenly, and the change of the absorption value within 3min at the wavelength of 210nm (the wavelength is 290nm when the substrate is tylosin) is recorded at the temperature of 30 ℃. The data from the three experiments were fitted using GraphPad Prism to obtain the enzymatic kinetic parameters as shown in the table (results are the average of 3 experiments). As can be seen from the table, the catalytic efficiency of the recombinase RmZHD-rHF is improved to a certain extent compared with that of the free enzyme RmZHD.
TABLE 1 metabolism kinetics of zearalenone degrading enzyme on erythromycin
Figure BDA0002328799960000071
TABLE 2 Metabolic kinetics of zearalenone degrading enzyme for tylosin
Figure BDA0002328799960000072
Sequence listing
<110> southern China university of agriculture
<120> application of zearalenone lactone hydrolase RmZHD in degradation of macrolide antibiotics
<141> 2019-12-20
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 268
<212> PRT
<213> McLeod beak (Rhinocladiella mackenziei CBS 650.93)
<400> 1
Met Ala Ala Thr Arg Thr Arg Gly Tyr Val Thr Thr Lys Asp Gly Ile
1 5 10 15
Lys Trp Tyr Tyr Glu Gln Glu Gly Ser Gly Pro Asp Val Val Leu Ile
20 25 30
Pro Asp Gly Leu Gly Glu Cys Gln Met Phe Asp Lys Pro Met Ser Leu
35 40 45
Ile Ala Ser Asn Gly Phe Arg Val Thr Thr Phe Asp Met Pro Gly Met
50 55 60
Ser Arg Ser Ser Asp Ala Pro Pro Glu Thr Tyr Gln Asp Ile Thr Gly
65 70 75 80
Arg Lys Leu Ala Gly Tyr Ile Ile Thr Leu Leu Asp Thr Leu Asp Ile
85 90 95
Lys Ile Ala Ser Val Trp Gly Cys Ser Ser Gly Ala Ser Thr Val Leu
100 105 110
Ala Leu Cys Ser Asp Tyr Pro Glu Arg Val Arg Asn Gly Met Pro His
115 120 125
Glu Val Pro Thr Glu Asn Pro Asp Ile Leu Leu His Ile His Glu Val
130 135 140
Asp Pro Ala Thr Ile Ser Gln Glu Met Ala Ala Asn Ser Arg Ala Tyr
145 150 155 160
Ser Gly Asn Val Glu Ala Trp Asp Ala Leu Gly Pro Glu Val His Ala
165 170 175
Arg Leu His Asp Asn Tyr Pro Arg Trp Ala Tyr Gly Tyr Pro Arg Thr
180 185 190
Ile Pro Pro Ser Ala Pro Val Lys Thr Glu Asp Leu His Lys Val Pro
195 200 205
Ile Asp Trp Thr Val Gly Ala Ser Thr Pro Thr Lys Leu Phe Phe Glu
210 215 220
Asn Ile Val Ile Ala Ala Arg Glu Gly Ile Asn Ile Gly Thr Leu Pro
225 230 235 240
Gly Asn His Phe Pro Tyr Val Ser His Pro Glu Glu Phe Ala Lys Tyr
245 250 255
Val Val Glu Thr Ser Arg Lys Tyr Leu Lys Gly Ser
260 265
<210> 2
<211> 804
<212> DNA
<213> McLeod beak (Rhinocladiella mackenziei CBS 650.93)
<400> 2
atggcagcca cccgtacccg cggctacgtt accaccaaag acggcatcaa gtggtactac 60
gagcaagaag gcagtggccc ggatgtggtt ctgattccgg atggtttagg cgagtgccag 120
atgttcgaca aaccgatgtc tttaattgcc agcaacggct ttcgcgtgac caccttcgat 180
atgccgggta tgagtcgcag tagcgatgca ccgccggaga cctatcaaga tattaccggc 240
cgcaaactgg ctggttatat catcacttta ctggatactt tagatatcaa gatcgccagc 300
gtgtggggtt gtagtagcgg tgcaagcacc gtgctggcac tgtgcagcga ttatccggaa 360
cgcgttcgca atggtatgcc gcatgaagtg ccgaccgaaa acccggacat tttactgcat 420
atccatgaag ttgatcccgc taccattagc caagaaatgg cagccaatag ccgcgcatac 480
agcggcaatg tggaagcatg ggatgcttta ggtccggaag tgcatgcacg tctgcatgat 540
aactatccgc gctgggcata tggctatcct cgcaccattc cgccgagcgc accggtgaaa 600
accgaggatc tgcacaaagt gccgatcgat tggaccgtgg gtgccagcac cccgaccaaa 660
ctgttttttg agaacattgt gattgcagcc cgtgaaggca tcaacattgg cactttaccg 720
ggtaatcatt ttccgtacgt gagccatccg gaggaattcg ccaaatacgt ggtggagacc 780
agccgtaaat atctgaaagg atcc 804
<210> 3
<211> 466
<212> PRT
<213> recombinant protease (RmZHD-rHF)
<400> 3
Met Ala Ala Thr Arg Thr Arg Gly Tyr Val Thr Thr Lys Asp Gly Ile
1 5 10 15
Lys Trp Tyr Tyr Glu Gln Glu Gly Ser Gly Pro Asp Val Val Leu Ile
20 25 30
Pro Asp Gly Leu Gly Glu Cys Gln Met Phe Asp Lys Pro Met Ser Leu
35 40 45
Ile Ala Ser Asn Gly Phe Arg Val Thr Thr Phe Asp Met Pro Gly Met
50 55 60
Ser Arg Ser Ser Asp Ala Pro Pro Glu Thr Tyr Gln Asp Ile Thr Gly
65 70 75 80
Arg Lys Leu Ala Gly Tyr Ile Ile Thr Leu Leu Asp Thr Leu Asp Ile
85 90 95
Lys Ile Ala Ser Val Trp Gly Cys Ser Ser Gly Ala Ser Thr Val Leu
100 105 110
Ala Leu Cys Ser Asp Tyr Pro Glu Arg Val Arg Asn Gly Met Pro His
115 120 125
Glu Val Pro Thr Glu Asn Pro Asp Ile Leu Leu His Ile His Glu Val
130 135 140
Asp Pro Ala Thr Ile Ser Gln Glu Met Ala Ala Asn Ser Arg Ala Tyr
145 150 155 160
Ser Gly Asn Val Glu Ala Trp Asp Ala Leu Gly Pro Glu Val His Ala
165 170 175
Arg Leu His Asp Asn Tyr Pro Arg Trp Ala Tyr Gly Tyr Pro Arg Thr
180 185 190
Ile Pro Pro Ser Ala Pro Val Lys Thr Glu Asp Leu His Lys Val Pro
195 200 205
Ile Asp Trp Thr Val Gly Ala Ser Thr Pro Thr Lys Leu Phe Phe Glu
210 215 220
Asn Ile Val Ile Ala Ala Arg Glu Gly Ile Asn Ile Gly Thr Leu Pro
225 230 235 240
Gly Asn His Phe Pro Tyr Val Ser His Pro Glu Glu Phe Ala Lys Tyr
245 250 255
Val Val Glu Thr Ser Arg Lys Tyr Leu Lys Gly Ser Gly Gly Gly Gly
260 265 270
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Thr Thr Ala Ser
275 280 285
Thr Ser Gln Val Arg Gln Asn Tyr His Gln Asp Ser Glu Ala Ala Ile
290 295 300
Asn Arg Gln Ile Asn Leu Glu Leu Tyr Ala Ser Tyr Val Tyr Leu Ser
305 310 315 320
Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu Lys Asn Phe Ala
325 330 335
Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg Glu His Ala Glu Lys
340 345 350
Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg Ile Phe Leu Gln Asp
355 360 365
Ile Lys Lys Pro Asp Cys Asp Asp Trp Glu Ser Gly Leu Asn Ala Met
370 375 380
Glu Cys Ala Leu His Leu Glu Lys Asn Val Asn Gln Ser Leu Leu Glu
385 390 395 400
Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro His Leu Cys Asp Phe
405 410 415
Ile Glu Thr His Tyr Leu Asn Glu Gln Val Lys Ala Ile Lys Glu Leu
420 425 430
Gly Asp His Val Thr Asn Leu Arg Lys Met Gly Ala Pro Glu Ser Gly
435 440 445
Leu Ala Glu Tyr Leu Phe Asp Lys His Thr Leu Gly Asp Ser Asp Asn
450 455 460
Glu Ser
465
<210> 4
<211> 59
<212> DNA
<213> human (Homo sapiens)
<400> 4
cgcggatccg gaggaggagg atcaggagga ggaggatcag gaggaggagg atcaatgac 59
<210> 5
<211> 44
<212> DNA
<213> human (Homo sapiens)
<400> 5
cccaagcttt tagctttcat tatcactgtc tcccagggtg tgct 44
<210> 6
<211> 37
<212> DNA
<213> McLeod beak (Rhinocladiella mackenziei CBS 650.93)
<400> 6
ctagctagca tggcagccac ccgtacccgc ggctacg 37
<210> 7
<211> 34
<212> DNA
<213> McLeod beak (Rhinocladiella mackenziei CBS 650.93)
<400> 7
cgcggatcct ttcagatatt tacggctggt ctcc 34

Claims (4)

1, SEQ ID NO: 1 in the formula (1), the zearalenone degrading enzyme RmZHD is applied to degrading macrolide antibiotics or preparing products for degrading macrolide antibiotics.
SEQ ID NO: 2, and the zearalenone degrading enzyme RmZHD coding gene is applied to the preparation of products for degrading macrolide antibiotics.
3, SEQ ID NO: 3, the recombinant enzyme RmZHD-rHF shown in the figure 3 is applied to degrading macrolide antibiotics or preparing products for degrading macrolide antibiotics.
4. Use according to claim 1 or 2 or 3, characterized in that the macrolide antibiotic is erythromycin and/or tylosin.
CN201911327678.XA 2019-12-20 2019-12-20 Application of zearalenone lactone hydrolase RmZHD in degradation of macrolide antibiotics Active CN111202940B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201911327678.XA CN111202940B (en) 2019-12-20 2019-12-20 Application of zearalenone lactone hydrolase RmZHD in degradation of macrolide antibiotics

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201911327678.XA CN111202940B (en) 2019-12-20 2019-12-20 Application of zearalenone lactone hydrolase RmZHD in degradation of macrolide antibiotics

Publications (2)

Publication Number Publication Date
CN111202940A CN111202940A (en) 2020-05-29
CN111202940B true CN111202940B (en) 2021-05-04

Family

ID=70782640

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201911327678.XA Active CN111202940B (en) 2019-12-20 2019-12-20 Application of zearalenone lactone hydrolase RmZHD in degradation of macrolide antibiotics

Country Status (1)

Country Link
CN (1) CN111202940B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114605557B (en) * 2022-03-10 2023-07-11 华南农业大学 Degradation aflatoxin B 1 And/or zearalenone fusion protein and preparation method and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Magnetic Resonance Visualization of Tumor Angiogenesis by Targeting Neural Cell Adhesion Molecules with the Highly Sensitive Gadolinium-loaded Apoferritin Probe;Geninatti-Crich S等;《Cancer Research》;20061231;第66卷(第18期);第9196-9201页 *
来源于 Rhinocladiella mackenziei 的玉米赤霉烯酮水解酶的表达纯化与酶学性质;胡翔颖等;《微生物学通报》;20181220;第45卷(第12期);第2585-2591页 *
玉米赤霉烯酮生物脱毒及关键酶作用机理的研究进展;唐语谦等;《现代食品科技》;20130715;第29卷(第7期);摘要，第1742页左栏倒数第1段-第1743页倒数第2段 *

Also Published As

Publication number Publication date
CN111202940A (en) 2020-05-29

Similar Documents

Publication Publication Date Title
CN113151069B (en) Bacillus subtilis and application thereof in preparation of antibacterial peptide and feed
Niu et al. Complete sequence and detailed analysis of the first indigenous plasmid from Xanthomonas oryzae pv. oryzicola
CN111961617A (en) Multi-effect bacillus subtilis for high yield of immune polysaccharide and bacteriocin and application thereof
Pilehchian et al. Fusion of Clostridium perfringens type D and B epsilon and beta toxin genes and it’s cloning in E. coli
CN110621775B (en) Novel pseudomonas aeruginosa phage Pse-AEP-4 and use thereof for inhibiting pseudomonas aeruginosa proliferation
CN111202940B (en) Application of zearalenone lactone hydrolase RmZHD in degradation of macrolide antibiotics
Leclercq et al. Plasmid-mediated resistance to lincomycin by inactivation in Staphylococcus haemolyticus
Nakamura et al. Macrolide esterase-producing Escherichia coli clinically isolated in Japan
CN113087771B (en) Penaeus vannamei DNA (deoxyribonucleic acid) combined antibacterial peptide VPDB40 and application thereof
CN106632606B (en) Antibacterial lipopeptide bacaucin derivative and application thereof in inhibiting bacterial infection
Zhang et al. A molecular mechanism of resistance to streptomycin in Xanthomonas oryzae pv. oryzicola
CN109112079B (en) Bacillus thuringiensis with bacteriostatic activity on several pathogenic bacteria and bacteriocin thereof
Wu et al. Genome-guided purification and characterization of polymyxin A1 from Paenibacillus thiaminolyticus SY20: a rarely explored member of polymyxins
Zidour et al. Genome sequencing and analysis of Bacillus pumilus ICVB403 isolated from Acartia tonsa copepod eggs revealed surfactin and bacteriocin production: insights on anti-Staphylococcus activity
WO2022141936A1 (en) Standard reference strains of staphylococcus argenteus containing specific molecular target and detection and use thereof
CN114891678A (en) Bacillus polymyxa CPL258 and screening and application thereof
Mirkovic et al. C-protein α-antigen modulates the lantibiotic thusin resistance in Streptococcus agalactiae
Shanmugaraju et al. Partial purification and characterization of Anti-MRSA peptide from marine Pseudomonas aeruginosa
CN115851525B (en) Chloramphenicol degrading bacterium, chloramphenicol dehydrogenase, encoding gene and application thereof
CN114605510B (en) Protein A10 with arsenite and methyl arsenite binding capacity, engineering strain containing protein gene and application
CN116003520B (en) Lactobacillus paracasei arginine biosynthesis protein antibacterial peptide NGJ1D and application thereof
CN116640755B (en) Streptococcus prophage lyase lys1519 and application thereof
CN114107271B (en) Heat-resistant and nutrient-resistant salmonella broad spectrum lyase with in-vitro cleavage activity, and preparation and application thereof
CN114480204B (en) Bacillus nicotinate FY2 and application thereof
CN118546916B (en) Enterotoxigenic escherichia coli phage, preparation and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant