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

Multi-copper oxidase, coding gene thereof, recombinant vector, recombinant strain and application

A technology for multi-copper oxidase and copper oxidase activity, applied in the field of enzyme engineering, can solve the problems of high degradation rate of aflatoxin, no measurement of actual degradation, and high cost

Inactive Publication Date: 2019-04-12
ANHUI AGRICULTURAL UNIVERSITY
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Dellafiora et al. discovered another laccase from a white-rot fungus, but only analyzed the possible interaction sites between laccase and AFB1 through 3D molecular modeling technology, and the actual degradation effect was not determined
In addition, laccases or other multi-copper oxidases from fungi are expressed in prokaryotic systems. Due to the preference of the expression system, the expression is difficult, the yield is high and the cost is low, and inclusion bodies are easy to form
Therefore, there is no high aflatoxin degradation rate at present, and is suitable for the multi-copper oxidase of industrial production

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
  • Multi-copper oxidase, coding gene thereof, recombinant vector, recombinant strain and application
  • Multi-copper oxidase, coding gene thereof, recombinant vector, recombinant strain and application
  • Multi-copper oxidase, coding gene thereof, recombinant vector, recombinant strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1: cDNA synthesis and cloning of multiple copper oxidase gene

[0055] The strain was derived from the Stenotrophomonas CW117 obtained in the laboratory in the early stage. Through gene sequence determination, the open reading frame nucleotide sequence of the multi-copper oxidase gene was analyzed, and the upstream primer of the primer that amplified the complete coding reading frame was designed ( LA-F) (SEQ ID NO.3): 5'-ATCTGGTTCCGCGT GGATCC ATGGCCGCCGCGTTGC-3'; downstream primer (LA-R) (SEQ ID NO.4): 5'-TCACGATGCGGCCG CTCGAG TCACCGCGCCATCCACAC-3', respectively introduce restriction endonuclease sites on the upstream and downstream primers (depending on the expression vector selected, BamHI and XhoI enzyme cutting sites have been added in the present invention, which have been underlined, The sequence in italics is the homologous sequence at the downstream end of the pGEX-4T-1 vector), and the polycopper oxidase gene encoding the amino acid shown in SEQ ...

Embodiment 2

[0056] Example 2: Heterologous Expression of Stenotrophomonas Multicopper Oxidase Gene

[0057] The E. coli BL21(DE3) / pGEX-4T-1 / Lac1 transformant obtained in Example 1 was cultured overnight on a shaker in 100 mL LB liquid medium containing 100 μg / mL ampicillin. Draw 1.0 mL of seed bacteria liquid and inoculate it into fresh 100 mL of LB liquid medium (containing 100 μg / mL ampicillin), and culture at 37° C. with shaking at 180 r / min. When the bacteria solution OD 600 When it reaches 0.6-0.8, add 0.2mmol / mL IPTG (isopropylthiogalactoside) to induce expression at 16°C for 4h. Refrigerate and centrifuge at 7000r / min for 10min, discard the supernatant. Suspend the cells with 10 mL of 1×phosphate buffered saline (PBS), and disrupt the cells by ultrasonic in an ice bath. The supernatant was collected by centrifugation, and after the supernatant was filtered, the expressed recombinant enzyme was purified by GST affinity chromatography to obtain pure enzyme. SDS-PAGE electrophores...

Embodiment 3

[0058] Example 3: Application of heterologous recombinant multi-copper oxidase in the degradation of aflatoxin AFB1

[0059] 1. Experimental materials

[0060] The enzyme preparation is a recombinant protein, and other reagents are analytically pure chemical reagents.

[0061] 2. Experimental method

[0062] Take 250 μL (1.0 mg / mL) of the purified recombinant multi-copper oxidase in Example 2 and mix it with an equal volume of 80 μg / L aflatoxin AFB1 (diluted working solution in PBS), incubate at 35°C for 24h, and incubate at 100°C in a water bath. The reaction was terminated by boiling for 10 min. Take 600 μL of the reacted samples and mix them with 900 μL of chromatographic grade acetonitrile, and remove the denatured protein by high-speed centrifugation and 0.22 μm membrane filtration, and then detect the remaining AFB1 concentration in the samples by UPLC. The blank control group was added with an equal amount of buffer for dissolving oxidase. It was found that the degr...

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
Theoretical molecular weightaaaaaaaaaa
Login to View More

Abstract

The invention discloses multi-copper oxidase, a coding gene thereof, a recombinant vector, a recombinant strain and an application, and belongs to the technical field of enzyme engineering. The invention provides the multi-copper oxidase, the coding gene thereof, the recombinant vector and the recombinant strain, and also provides an application of the multi-copper oxidase to degradation of aflatoxins AFB1 and biological toxicity removal in food grains and fermented tea leaves. The testing result indicates that when the multi-copper oxidase treats AFB1 for 24h in a buffer system with pH being7.0 under the condition of 37 DEG C, the degradation rate of the AFB1 reaches 44%.

Description

technical field [0001] The invention relates to a multi-copper oxidase and its coding gene, recombinant carrier, recombinant bacteria and application, and belongs to the technical field of enzyme engineering. Background technique [0002] Aspergillus mycotoxins are mainly a class of secondary metabolites produced by toxin-producing Aspergillus strains such as Aspergillus flavus, Aspergillus parasiticus and ochratoxin. More than 20 structural analogues have been isolated and identified, among which aflatoxin B1 (AFB1) and Ochratoxin A (OTA) is the most toxic and most widely polluted in agricultural production and food industry. In 1993, the Cancer Research Institute of the World Health Organization (WHO) classified aflatoxin as a class I carcinogen and ochratoxin as a class IIB carcinogen. Because aflatoxin has strong carcinogenic, teratogenic and mutagenic properties, it has gradually become the focus of public health and scientific research. Aflatoxin contamination of gra...

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): C12N9/02C12N15/53A23L5/20
CPCA23L5/25C12N9/0061C12Y110/03002
Inventor 周育陈楠蔡梦宇祁克宗王旭
Owner ANHUI AGRICULTURAL UNIVERSITY
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