Thermophilic esterase and application thereof in degradation of PAEs (Phthalic Acid Esters)

A technology of esterase and biological enzyme, applied in the field of bioengineering, can solve the problems of little practical application value and poor stability, and achieve the effects of superior thermal stability, fast reaction speed and good stability of degradation reaction

Inactive Publication Date: 2014-11-19
EAST CHINA UNIV OF SCI & TECH
View PDF2 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These enzymes are all isolated from mesophilic microorganisms with a single ecolog

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
  • Thermophilic esterase and application thereof in degradation of PAEs (Phthalic Acid Esters)
  • Thermophilic esterase and application thereof in degradation of PAEs (Phthalic Acid Esters)
  • Thermophilic esterase and application thereof in degradation of PAEs (Phthalic Acid Esters)

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0045] Example 1 Plasmid construction of thermophilic esterase gene

[0046] Using the Sulfobacillus acidophilus genome as a template, 10332-Nde IF and 10332-HindIII-R were used as forward and reverse primers to PCR amplify the 10332 gene fragment encoding PAEs hydrolase with a size of about 900 bp. The PCR product was in agar Carbohydrate gel electrophoresis inspection. The PCR product obtained was ligated into pET28a vector and transformed into E. coli DH5α, and multiple white colonies were selected for identification. Through bacterial electrophoresis screening, the plasmid was extracted for double enzyme digestion verification, and the correct pET28a-10332 recombinant plasmid containing the target gene was obtained, and the recombinant plasmid was transformed and stored in E. coli BL21 (DE3). After sequencing, it was correct and successfully constructed The recombinant plasmid pET28a-10332 which can express the target gene 10332 heterologously and its expression transformant....

Example Embodiment

[0105] Example 3 Heterologous expression and separation and purification of recombinant E. coli

[0106] The expression transformant BL21(DE3) / pET28-10332 is cultivated, and the target protein can be soluble expression through the appropriate temperature and appropriate concentration of the inducer IPTG, so that the enzyme can be prepared in large quantities. The induced expression of the bacterial cell was disrupted by ultrasound, and the supernatant after ultrasound was separated and purified by histidine tag affinity chromatography to obtain electrophoresis pure protein with his-tag tag thermophilic esterase.

[0107] 1 Induced expression of foreign fragments in E. coli

[0108] After the expression transformant BL21(DE3) / pET28-10332 was cultured on a 37°C constant temperature plate for 12 hours, a single colony was picked and placed in 5ml LB (containing 50μg / ml kanamycin) liquid medium. After 8 hours of culture, make glycerol Manage and protect the species for use. Take 5μl of...

Example Embodiment

[0129] Example 4 Enzymatic properties and substrate profile of recombinant E. coli expression products

[0130] Protein concentration detection and enzyme activity detection were performed on the obtained crude protease solution and purified protein. The specific activity of the enzyme was very high, up to 2600 U / mg. Using pNP esters with different chain lengths, the reaction efficiency of the enzyme for different substrates was measured, and the reaction substrate spectrum of the enzyme and the best substrate of the enzyme for pNP esters were obtained. Test the resistance of the enzyme to heat, denaturants, organic solvents and metal ions. The enzyme has good thermal stability, high resistance to low concentrations of urea and Tween, and is resistant to organic solvents and metal ions. General tolerance. Through amino acid alignment analysis and conservative sequence analysis, the highest similarity between this enzyme and other enzymes was only 52%, and it was determined to be...

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

No PUM Login to view more

Abstract

The invention discloses a thermophilic esterase and an application thereof in degradation of PAEs (Phthalic Acid Esters). The amino acid sequence of the thermophilic esterase is shown in SEQ ID NO.1. A hydrolysis reaction of PAEs is carried out in a water-containing medium under the catalysis of the thermophilic esterase and the fusion protein of the thermophilic esterase so as to generate a phthalic monomer acid and an alcohol are generated, and a phthalic acid and the alcohol are further formed. The thermophilic esterase can be used for preliminarily degrading a plasticizer, has a certain resistance to a denaturant and an organic solvent with moderate and low concentrations, has excellent heat stability and has the function of degrading various PAEs. The thermophilic esterase is the only one esterase cloned from thermophilic microorganisms according to literature reports. The thermophilic esterase has a good application prospect in the purely enzymatic degradation of the PAEs on the basis of the excellent performance. The thermophilic esterase realizes harmless degradation of the PAEs, and is good in degradation reaction stability, extensive in reaction condition and high in reaction speed.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a thermophilic esterase and its application in degrading PAEs. Background technique [0002] Various chemical synthetic substances play a vital role in human life, they improve people's life and promote the development of human social civilization. But at the same time, they also pollute our earth and bring great pressure to our living environment. There are also some compounds whose harm to the environment has been verified after years of scientific experiments. At this time, their accumulation range and accumulation amount in nature have reached a serious level. Phthalate esters (PAEs), also known as phthalates, are a general term for about 30 compounds that are widely used in daily life and are mainly used as plasticizers for plastic products. They are reproductively toxic at very low concentrations , and after it enters the environment, it is not easy to degrade rapi...

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): C12N9/18C12N15/55C12N15/63C12N1/21A62D3/02C12R1/19C12R1/01A62D101/28
CPCA62D3/02A62D2101/28C07K2319/21C12N9/18
Inventor 范翔张晓彦李骋远邢帅许建和
Owner EAST CHINA UNIV OF SCI & TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap