Supercharge Your Innovation With Domain-Expert AI Agents!

A method for improving enzyme stability

An alkaline, pectinase technology, applied in the direction of enzyme stabilization, lyase, hydrolase, etc., can solve the problem of difficulty in obtaining thermostable mutants, and achieve the effect of improving stability

Active Publication Date: 2021-08-24
JIANGNAN UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the above-mentioned molecular transformation technology has become a routine strategy for enzyme thermostability transformation, it still has its inherent technical defects. For example, the premise of site-directed mutation is to obtain accurate molecular structure information of the enzyme, while in vitro directed evolution faces a large number of mutations. Mutants with significantly improved thermal stability are difficult to obtain in a short period of time

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 for improving enzyme stability
  • A method for improving enzyme stability
  • A method for improving enzyme stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Embodiment 1: the construction of the recombinant bacterium that can express fusion enzyme

[0104] Specific steps are as follows:

[0105] Construction of recombinant bacteria that can express fusion alkaline pectinase:

[0106] Using pET22b(+) as the plasmid backbone, the gene of lipoxygenase (LOX) was cloned between Nco I and Xho I to obtain the recombinant plasmid pET22b(+) / lox expressing wild LOX, and the coding of SAP and linker The gene was inserted between Nde I and Nco I of pET22b(+) / lox (such as figure 1 shown), the plasmids pET22b(+) / sap1-L1-lox and pET22b(+) / sap2-L2-lox expressing LOX fusion enzymes SAP1-L1-LOX and SAP2-L2-LOX were respectively obtained and transferred into Expression host Escherichia coli (Escherichia coli) BL21 (DE3) (SAP1-L1-LOX comprises the lipoxygenase of amino acid sequence as shown in SEQ ID NO.5 and the linker linking by amino acid sequence as shown in SEQ ID NO.6 The amino acid sequence at the lipoxygenase N-terminal is SAP show...

Embodiment 2

[0117] Embodiment 2: the preparation of fusion enzyme and wild enzyme

[0118] Specific steps are as follows:

[0119] Picking the single bacterium colony of the recombinant bacterium that embodiment 1 obtains is inserted in the Erlenmeyer flask (250mL) of 25mL, 37 ℃ of culture temperature, shaker speed 200r / min, cultivate 12h, obtain seed liquid; Press 3% Inoculum size Put the seed solution into a conical flask (250mL) with a liquid volume of 25mL, cultivate at a temperature of 37°C, and when the OD 600 When it reaches 0.6, add IPTG induction (wherein the induction amount of LOX is 1 mM, PGL is 0.04 mM, and ASN is 1 mM)), and at the same time adjust the temperature to the most suitable induction temperature for the enzyme and cultivate (wherein, LOX is 20 ° C for 24 h, PGL was cultured at 30°C for 48h, and ASN was cultured at 30°C for 12h), to obtain fusion enzymes containing different fusion enzymes SAP1-L1-LOX, SAP2-L2-LOX, SAP1-L1-PGL, SAP2-L2-PGL, SAP1-L1-ASN, Fermentat...

Embodiment 3

[0120] Embodiment 3: the impact of different stabilizers on fusion enzyme and wild enzyme

[0121] Specific steps are as follows:

[0122] The different fusion enzymes SAP1-L1-LOX, SAP2-L2-LOX, SAP1-L1-PGL, SAP2-L2-PGL, SAP1-L1-ASN, SAP2-L2-ASN and wild enzyme LOX obtained in Example 2, The fermentation broth of PGL and ASN is purified to obtain pure enzyme liquid; with the pure enzyme liquid without adding any stabilizer as a contrast, after adding different types and concentrations of stabilizers in the pure enzyme liquid as shown in Table 1-3, the The pure enzyme solution of the control group (without stabilizer added) and the experimental group (with stabilizer added) was tested for thermal stability (the test results were as follows: Figure 2-10 ).

[0123] Table 1 Types and concentrations of stabilizers added to LOX pure enzyme solution

[0124] stabilizer concentration Polyethylene glycol octylphenyl ether (Triton X-100) 0.01% (v / v) Tween (Tw...

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 method for improving enzyme stability and belongs to the technical field of enzymes. The method of the present invention greatly improves the stability of the enzyme by linking the amphipathic short peptide (SAP) at the N-terminus of the enzyme; the lipoxygenase prepared by the method of the present invention remains stable after being incubated at 50°C for 30min. Maintain more than 95% of the initial enzyme activity, while wild-type LOX only retains about 20% of the initial enzyme activity under the same conditions; the alkaline pectinase prepared by this method still maintains 97% of the initial enzyme activity after being incubated at 60°C for 30 minutes Above, but under the same conditions, wild-type LOX only retains about 35% of the initial enzyme activity; the asparaginase prepared by this method can maintain more than 120% of the initial enzyme activity after being incubated at 60°C for 30 minutes, while the wild-type LOX under the same conditions Type LOX only retains about 30% of the initial enzyme activity.

Description

technical field [0001] The invention relates to a method for improving enzyme stability, which belongs to the technical field of enzymes. Background technique [0002] Based on the important influence of thermostability on the application performance of enzymes, obtaining enzymes with high thermostability has always been a research hotspot in the field of enzyme engineering. [0003] At present, with the development of structural biology and bioinformatics, researchers have been able to more accurately locate the thermal stability of enzyme molecules through the analysis of some structural parameters (such as B-factor, RMSF value, etc.) or homologous sequence comparison. Specific amino acid residues or peptides, and then conduct site-directed mutations to improve the thermal stability of the enzyme. [0004] Although the above-mentioned molecular transformation technology has become a routine strategy for enzyme thermostability transformation, it still has its inherent tech...

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): C12N9/96C12N9/18C12N9/24C12N9/88C12N9/82C12N9/02
Inventor 刘松庞翠萍陈坚堵国成赵伟欣
Owner JIANGNAN UNIV
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

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

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com