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Application of zearalenone lactone hydrolase RmZHD in degradation of macrolide antibiotics

A technology of zearalenone and macrolides, which is applied in the biological field, achieves great application prospects, improves the activity and expression of enzymes, and reduces antibiotic pollution.

Active Publication Date: 2020-05-29
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Hu Xiangying et al. (2018) disclosed a zearalenone hydrolase RmZHD derived from Rhinocladiella mackenziei (Hu Xiangying, Liu Wenting, Liu Weidong, et al. Expression, purification and enzymatic properties of zearalenone hydrolase derived from Rhinocladiella mackenziei [J]. Microbiology Bulletin, 2018,45(12):51-57.); However, there is no report on the application of the zearalenone hydrolase RmZHD in the degradation of macrolide antibiotics

Method used

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  • Application of zearalenone lactone hydrolase RmZHD in degradation of macrolide antibiotics
  • Application of zearalenone lactone hydrolase RmZHD in degradation of macrolide antibiotics
  • Application of zearalenone lactone hydrolase RmZHD in degradation of macrolide antibiotics

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] In vitro expression of embodiment 1 recombinant enzyme RmZHD-rHF and free enzyme RmZHD

[0034] 1. Expression vector construction

[0035] Fusion protein design strategy: Human ferritin (rHF) was used as a fusion partner, and zearalenone degrading enzyme (RmZHD) was placed at the N-terminus of rHF for expression, and a linker was used between RmZHD and rHF connect.

[0036] Construction of the expression vector: According to the sequences of RmZHD and pET28a-rHF, specific amplification primers (Table 1) were designed, Nhe I and BamH I restriction sites were respectively introduced at both ends of the RmZHD gene, and a large amount of RmZHD sequences were amplified ( Use primers 3 and 4 to amplify the RmZHD sequence for constructing the pET28a-RmZHD vector, and utilize primers 3 and 5 to amplify the RmZHD sequence for constructing the pET28a-RmZHD-rHF vector); introduce the core of the linker (GGGGS) at the 5' end of the rHF gene Nucleotide sequence, while introducing ...

Embodiment 2

[0049] Example 2 Recombinase RmZHD-rHF and Free Enzyme RmZHD Antibiotic Degradation Activity Test

[0050] The growth curve was used to test the degradation activity of the recombinant enzyme RmZHD-rHF and the free enzyme RmZHD on macrolide antibiotics (e.g. erythromycin and tylosin).

[0051] (1) Determine the minimum inhibitory concentration of erythromycin through the drug susceptibility test (MIC).

[0052] (2) Cultivate Bacillus (ATCC 29213) at a drug concentration of 1 / 2MIC, use LB broth as a control, add antibiotics, antibiotics after RmZHD treatment, antibiotics after RmZHD-rHF treatment, RmZHD and RmZHD-rHF as experiments Group.

[0053] (3) Inoculate bacteria at a ratio of 1:100, culture at 37°C, 200rpm, measure OD every 1 hour 600 , record the measured value.

[0054] (4) The determination method of the growth curve of tylosin is the same as that of erythromycin.

[0055] Results: The growth curve of Bacillus treated with erythromycin was as follows image 3 As...

Embodiment 3

[0056] Enzyme activity assay of embodiment 3 recombinase RmZHD-rHF and free enzyme RmZHD

[0057] By using a Shimadzu UV-2550 spectrophotometer, a full-wavelength scan of erythromycin, tylosin and enzyme-treated products was detected. After scanning, it was found that after the degradation by zearalenone degrading enzyme, a specific ultraviolet absorption peak appeared at 210nm, and the metabolic kinetics of zearalenone degrading enzyme to erythromycin was determined by measuring the increase of the product at 210nm. Tylosin was scanned and found that the specific absorption peak at 290nm decreased, and the metabolic kinetics of zearalenone degrading enzyme to tylosin was determined by measuring the decrease of the substrate at 290nm.

[0058] The total volume of the standard reaction system is 800μL, which contains 50nM RmZHD-rHF or RmZHD and different concentrations of erythromycin (the enzyme concentration is 10nM when the substrate is tylosin), and the buffer is 25mM Tris-...

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Abstract

The invention discloses an application of zearalenone lactone hydrolase RmZHD in degradation of macrolide antibiotics. Research finds that the zearalenone hydrolase RmZHD can efficiently degrade macrolide antibiotics, and can be used for reducing antibiotic pollution in the environment. The invention also provides a recombinase RmZHD-rHF containing the zearalenone hydrolase RmZHD. The activity andexpression quantity of the enzyme of RmZHD can be further improved, and the application prospect is great.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, relates to the application of zearalenone lactone hydrolase RmZHD in degrading macrolide antibiotics. Background technique [0002] Antibiotics are widely used in the prevention and treatment of diseases in animal husbandry and the prevention and control of animal and plant diseases and insect pests. Improper use of antibiotics or illegal excessive addition can cause their residues in animals. After antibiotics enter animals or humans, only a small part is metabolized and absorbed, but more than half of antibiotics are digested and absorbed by organisms and excreted in the form of parent or metabolites through animal urine and feces. [0003] Macrolide antibiotics are classic antibiotics commonly used in clinical practice, and their molecules have a macrolide structure, and most of them are basic lipophilic compounds. Among them, Tylosin is mainly used clinically for the treatmen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A62D3/02C12N9/16C07K19/00C12N15/70A62D101/28
CPCA62D3/02A62D2101/28C07K2319/735C12N9/16C12N15/70
Inventor 文继开孙天钰邓诣群吴骏陈庆梅母培强邓凤如
Owner SOUTH CHINA AGRI UNIV
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