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

Homoserine lactonase mutant, coding gene and application of homoserine lactonase mutant in replacing antibiotics

A technology of homoserine lactone and serine lactone, applied in the field of agricultural biology, can solve the problems of poor enzyme stability and high use cost, achieve the effects of improving specific activity, reducing use cost, and meeting application and promotion needs

Active Publication Date: 2020-10-02
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, although the biological efficacy of N-acyl homoserine lactonase has been widely recognized, the enzyme has not been widely accepted and used on a large scale in the industry. The main reasons are that the stability of the enzyme is relatively poor, and the cost of use too high

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
  • Homoserine lactonase mutant, coding gene and application of homoserine lactonase mutant in replacing antibiotics
  • Homoserine lactonase mutant, coding gene and application of homoserine lactonase mutant in replacing antibiotics
  • Homoserine lactonase mutant, coding gene and application of homoserine lactonase mutant in replacing antibiotics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1 homoserine lactonase AIIA1 and mutants AIIA1-Y128H Cloning and expression vector construction

[0028] The present invention is derived from thermoacidophilic bacteria Alicyclobacillus acidoterrestris The homoserine lactonase (whose amino acid sequence is as shown in SEQ ID NO: 3) is the parent, which is expressed after replacing the Y128H site of the acidic homoserine lactonase using molecular biology techniques.

[0029] SEQ ID NO:3 is shown.

[0030] by Alicyclobacillus acidoterrestris Using genomic DNA as a template, the wild-type gene was first cloned and connected to the pET30a(+) expression vector to obtain AIIA1 -pET30a(+), with AIIA1 -pET30a(+) plasmid was used as a template, and the gene encoding a high specific activity homoserine lactonase mutant was amplified by over-lap PCR to construct AIIA1-Y128H - pET30a(+) expression vector.

Embodiment 2

[0031] Example 2 Preparation of Homoserine Lactonase Mutant with High Specific Activity.

[0032] Separately AIIA1 -pET30a(+) and AIIA1-Y128H - pET30a(+) expression vector was transformed into Escherichia coli competent cell BL21(DE3) to obtain a recombinant expression strain AIIA1 -pET30a(+)-BL21(DE3) and AIIA1-Y128H -pET30a(+)-BL21(DE3).

[0033] The constructed recombinant expression strain AIIA1 -pET30a(+)-BL21(DE3) and AIIA1-Y128H -pET30a(+)-BL21(DE3) was activated overnight, then transferred to a 1 L large bottle containing 300 ml LB culture medium (containing 50 μg / ml Kana) at 1%, and cultivated to OD at 37 °C and 220 rpm 600 When the value is about 0.6-0.8 (about 2-3 h), add a final concentration of 0.6 mM IPTG to induce expression, and transfer to 28 ° C, 190 rpm to induce culture for about 8-10 h. Collect bacteria by centrifugation at 12,000 rpm for 10 min. Bacteria were resuspended with 0.05mM crude enzyme solution at pH 7.0 and purified using His-tag p...

Embodiment 3

[0034] Example 3 Activity Analysis of Recombinant High Specific Activity Homoserine Lactonase Mutant and Wild Type

[0035] Enzyme activity was determined by HPLC method

[0036] (1) Principle of the method:

[0037] The quenching enzyme can break the lactone bond of N-acyl homoserine lactone to generate N-acyl homoserine, and the reduction of the substrate is proportional to the activity of the quenching enzyme in the reaction solution. Therefore, the activity of the quenching enzyme in the reaction solution can be calculated by measuring the reduction of the substrate by HPLC.

[0038] (2) Substrate solution: octanoyl homoserine lactone [3-oxo-C8-HSL] at a concentration of 0.1 mg / mL. Reaction system: draw 0.2ml of appropriately diluted enzyme solution, add 0.2ml of the above substrate, react at 30°C for 30 minutes, add 0.1ml of 10% SDS to terminate the reaction, and measure the remaining amount of the substrate.

[0039] (3) Mobile phase solution: 36% acetonitrile-water s...

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 relates to the technical field of agricultural biology, in particular to a homoserine lactonase mutant, a coding gene and an application of the homoserine lactonase mutant in replacing antibiotics. The specific structure of an enzyme molecule is modified to obtain the homoserine lactonase mutant with high specific activity, so that the purpose of improving the specific activity is achieved. The recombinant high-specific-activity homoserine lactonase mutant has the optimum pH value of 6.5 and the optimum temperature of 45 DEG C, which are consistent with those of a wild type, butthe specific activity is improved by 120% compared with that of the wild type, the use cost can be remarkably reduced, and the application and popularization requirements of industrial production andfeed enzyme preparations are better met.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a homoserine lactonase mutant, a coding gene and the application thereof to replace antibiotics. Background technique [0002] For a long time, the abuse of antibiotics in feed has brought serious food safety problems, and the research on antibiotic substitutes has been paid more and more attention. Seeking new biological agents with the function of replacing feed antibiotics has become a new research hotspot. N-acyl homoserine lactonase (quencher enzyme) can adopt the quorum sensing quenching strategy to prevent and control the pathogenic bacteria by destroying the quorum sensing system of the pathogenic bacteria. The regulation of the behavior of many microbial populations is mainly through the synthesis, secretion, and detection of the concentration of small signal molecules to sense the population density and regulate the expression of specific genes. This phenomeno...

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): C12N9/18C12N15/55C12N15/70C12N1/21C12P13/04A23K20/189A61K38/46A61P31/00C12R1/19
CPCA61K38/00A23K20/189A61P31/00C12N9/18C12N15/70C12P13/04C12Y301/01081
Inventor 柏映国黄火清姚斌罗会颖涂涛王亚茹苏小运王苑张杰师霞
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