A method of adding disulfide bonds to improve the thermostability of ferulic acid esterase a

A technology of ferulic acid esterase and nucleotide, applied in the field of bioengineering, can solve the problems of poor thermal stability of natural ferulic acid esterase, achieve large industrial production and application potential and economic value, improve thermal stability, The effect of improving thermal stability

Active Publication Date: 2017-06-13
JIANGNAN UNIV
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research results show that the thermal stability of natural ferulic acid esterase is generally poor, which becomes the bottleneck of its application in many fields such as food processing, feed (granulation), paper industry (pulp bleaching) and so on.

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
  • A method of adding disulfide bonds to improve the thermostability of ferulic acid esterase a
  • A method of adding disulfide bonds to improve the thermostability of ferulic acid esterase a
  • A method of adding disulfide bonds to improve the thermostability of ferulic acid esterase a

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Mutant gene AufaeA A126C-N152C and its expression plasmid construction

[0021] Using large primer PCR technology to construct the fusion gene, it is mainly divided into four steps: use 126-F, 152-R as primers for the first round of PCR (94°C for 5min; 2 cycles, 94°C for 30s, 45°C for 30s, 72°C for 15s ; 28 cycles, 94°C for 30s, 55°C for 30s, 72°C for 15s; 72°C for 10min; 10°C for storage) to obtain the gene fragment AufaeA 126-152 ; With pUCm-T-AufaeA as a template, the first round of PCR product AufaeA 126-152 Carry out the second round of large primer PCR (94°C for 5min; 2 cycles, 94°C for 30s, 45°C for 30s, 72°C for 30s) for the primer and the specific primer FAE-F in the applied invention patent (application number: 201210181372.X). 28 cycles, 94°C for 30s, 55°C for 30s, 72°C for 30s; 72°C for 10min; 10°C for preservation), the gene fragment AufaeA was obtained F-152 ; With pUCm-T-AufaeA as a template, the second round of PCR product AufaeA F-152 The ...

Embodiment 2

[0023] Embodiment 2GS115 / AufaeA A126C-N152C Construction, expression and determination of properties of recombinants

[0024] pPIC9K-AufaeA with SalI A126C-N152C Perform linearization, perform electrotransformation and screening according to the Pichia expression manual, and obtain high-copy Pichia recombinant GS115 / AufaeA A126C-N152C . The engineered bacteria were induced with 1.0% methanol for 72 hours. The centrifuged supernatant is the recombinant ferulic acid esterase A crude enzyme solution, which is detected as a single band by SDS-PAGE, and the recombinant AuFaeA is transformed A126C-N152C The optimum reaction temperature is 50°C, which is 5°C higher than that of the original enzyme. The half-life at 60°C is 40 minutes, and the thermal stability is also greatly improved compared with the original enzyme.

[0025]

[0026]

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 aims to provide a method for reforming heat stability of ferulic acid esterase A, constructing a mutant ferulic acid esterase A engineering bacterium and realizing high-efficiency heterologous expression of mutant ferulic acid esterase A. According to the space structure design mutant A126C and N152C derived from A.usamii E001 ferulic acid esterase A (AuFaeA), mutant ferulic acid esterase A: AuFaeA A126C-N152C is obtained, the mature peptide gene sequence thereof is SEQ ID NO:1, and C126 and C152 can form one disulfide bond. Experimental results indicate that the heat stability of the enzyme is obviously improved after mutation, thus providing a new technological path for reforming of other enzymes. As a heat-resistant enzyme preparation, the ferulic acid esterase has great industrial production potential and economic value.

Description

technical field [0001] The present invention relates to thermostability modification of Aspergillus usamii E001 strain ferulic acid esterase A (AuFaeA) gene, construction of mutant ferulic acid esterase A engineering bacteria and high-efficiency expression of recombinant mutant ferulic acid esterase A , belonging to the field of bioengineering technology. Background technique [0002] Ferulic acid esterase (EC 3.1.1.73), also known as cinnamic acid esterase, is a subclass of carboxylic acid hydrolase and belongs to extracellular enzymes, which can break the gap between hemicellulose and between hemicellulose and lignin. The ester bond between the crosslinks is one of the components of the hemicellulose degrading enzyme system. Since Faulds first isolated ferulic acid esterase in 1991, more than 30 ferulic acid esterases have been cloned, expressed and purified. Their main biological function is to hydrolyze the ester bond between polysaccharides and ferulic acid in plant ce...

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
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N9/18C12N15/66C12N15/81C12R1/84
Inventor 邬敏辰殷欣余涛李剑芳姚瑶何瑶
Owner JIANGNAN UNIV
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