Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Zearalenone hydrolase mutant ZHDM1, and encoding gene and application thereof

A technology of zearalenone and ZHDM1, applied in the field of agricultural biology, can solve the problems of low activity and hinder large-scale industrial production and application of zearalenone hydrolase, and achieve the effect of broad application prospects

Active Publication Date: 2019-12-13
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
View PDF4 Cites 11 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the activities of zearalenone hydrolase found so far are all low, which hinders the large-scale industrial production and application of zearalenone hydrolase

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Zearalenone hydrolase mutant ZHDM1, and encoding gene and application thereof
  • Zearalenone hydrolase mutant ZHDM1, and encoding gene and application thereof
  • Zearalenone hydrolase mutant ZHDM1, and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Preparation of recombinant strain X33 (pPICZ(α)A-zh607) containing wild-type erythralenone hydrolase gene

[0032] 1.1 Amplify the nucleic acid sequence zh607 of the wild-type ZH607 of the zearalenone hydrolase protein

[0033] After codon optimization, the coding gene zh607 of erythralenone hydrolase ZH607 was obtained. The PCR method was used to amplify the zh607 gene and the vector pPICZ(α)A nucleic acid fragment, and connect the two through a recombination kit to obtain the recombinant plasmid pPICZ(α)A-zh607, and transform Pichia pastoris X33 to obtain recombinant Pichia pastoris Strain X33 (pPICZ(α)A-zh607).

[0034] The primers used in PCR are as follows:

[0035] Table 1 Wild-type zearalenone hydrolase and specific primers for vector amplification

[0036]

[0037] Among them, zh607-pPICZ(α)A-F and zh607-pPICZ(α)A-R are used to amplify the gene coding sequence of wild type ZH607 of zearalenone hydrolase; pPICZ(α)A-zh607-F and pPICZ(α)A -zh607-R ...

Embodiment 2

[0042] Example 2 Preparation of recombinant strain X33 (pPICZ(α)A-zh607-AH) containing mutant zearalenone hydrolase gene

[0043] 2.1 Construction of recombinant plasmid pPICZ(α)A-zh607-AH

[0044] The optimized mutation site was designed to replace ASVTGME at position 136-142 with DILLHIH, and the mutation site was introduced by the method of point mutation kit, and it was sequenced and verified to obtain the chiaralenone hydrolase mutant plasmid pPICZ(α) A-zh607-AH. The primers used are shown in Table 2:

[0045] Table 2 Specific primers for mutant zearalenone hydrolase

[0046]

[0047] 2.2 Construction of recombinant strain X33 (pPICZ(α)A-zh607-AH)

[0048] The recombinant plasmid pPICZ(α)A-zh607-AH was digested with SacI, and the recovered product was transformed into Pichia pastoris competent cells X33 by electroporation to induce expression, and the recombinant expression strain X33 (pPICZ(α)A-zh607-AH) was obtained.

Embodiment 3

[0049] Example 3 Obtaining wild-type ZH607 and mutant ZHDM1 of erythralenone hydrolase protein

[0050] 3.1 Induced expression of protein ZH607 and ZHDM1

[0051] The obtained recombinant expression strains X33 (pPICZ(α)A-zh607) and X33(pPICZ(α)A-zh607-AH) were inoculated into YPD medium for seed culture, 200rpm, 30°C for 48h, and 1% The inoculum was transferred to BMGY medium, cultured at 200 rpm at 30°C for 48 hours, and after enough bacteria were enriched, the bacteria were collected and added to BMMY medium containing 1% methanol to induce expression.

[0052] 2. Purification of protein ZH607 and ZHDM1

[0053] Centrifuge the induced bacterial solution at 12,000 rpm for 10 minutes, collect the supernatant and concentrate it, then dialyze it with 10 mM disodium hydrogen phosphate solution (adjust the pH to 7.6 with citric acid), and then carry out ion exchange chromatography on the dialyzed enzyme solution. Solution A is 10mM disodium hydrogen phosphate solution (adjust t...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Specific vitalityaaaaaaaaaa
Specific vitalityaaaaaaaaaa
Login to View More

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to a zearalenone hydrolase mutant ZHDM1, and an encoding gene and an application thereof. The zearalenonehydrolase mutant ZHDM1 is obtained by mutating 136-142 amino acids of wild zearalenone hydrolase ZH607. The enzyme activity of the zearalenone hydrolase mutant ZHDM1 disclosed by the invention is 2.9times that of the wild type, so that the high-enzyme-activity mutant disclosed by the invention can meet the requirements on the zearalenone degradation activity in the fields of energy sources, foods, feeds and the like, and has a wide application prospect.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a mutant ZHDM1 of zearalenone hydrolase and its coding gene and application. Background technique [0002] Zearalenone (ZEN), also known as F-2 toxin, is a non-steroidal, estrogen-like mycotoxin produced by a variety of Fusarium species. The estrogenic toxicity of ZEN is not only manifested in the damage to the reproductive ability of female animals, but also can cause damage to the reproductive system of male animals. In addition, high-dose ZEN also has liver and kidney toxicity, intestinal toxicity, immunotoxicity and the combined toxicity of mycotoxins, which seriously endanger animal health. [0003] The microbial degradation method to eliminate ZEN pollution is the process of using the enzymes produced by microorganisms in the metabolic process to interact with ZEN to destroy the toxic groups of ZEN molecules, so that they can be degraded or converted i...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N9/14C12N15/55C12N15/81C12N1/19A23L5/20C12R1/84
CPCA23L5/25C12N9/14C12N15/815
Inventor 姚斌罗会颖于心蕊涂涛柏映国黄火清苏小运王亚茹王苑张杰
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com