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Enzymatic thermal stability improving method

A thermostability, recombinant enzyme technology, applied in the direction of chemical instruments and methods, biochemical equipment and methods, enzymes, etc., can solve the problems of inappropriate thermal stability transformation of enzyme molecules, etc., to improve the thermal stability of industrial enzymes, improve Thermal stability, easy to promote the effect

Active Publication Date: 2014-04-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods are not suitable for the thermostability modification of all enzyme molecules

Method used

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  • Enzymatic thermal stability improving method
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  • Enzymatic thermal stability improving method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Amino acid sequence of amphipathic short peptide and cloned into plasmid pET-22b(+)

[0024] 1 AEAEAKAKAEAEAKAK

[0025] 2. VNYGNGVSCSKTKCSVNWGQAFQERYTAGTNSFVSGVSGVASGAGSIGIGRR

[0026] 3. DWLKAFYDKVAEKLKEAFKVEPLRADWLKAFYDKVAEKLKEAF

[0027] 4. DWLKAFYDKVAEKLKEAFGLLPVLEDWLKAFYDKVAEKLKEAF

[0028] 5. DWLKAFYDKVAEKLKEAFKVQPYLDDWLKAFYDKVAEKLKEAF

[0029] 6. DWLKAFYDKVAEKLKEAFNGGARLADWLKAFYDKVAEKLKEAF

[0030] According to the above amino acid sequence, the DNA sequence was chemically synthesized, and cloned into the plasmid pET-22b(+) until the plasmid pET-22b(+) / AP

Embodiment 2

[0031] Embodiment 2: Construction of recombinant plasmid pET-22b(+) / AP-enzyme

[0032] The target enzyme gene was cloned into the Nco I and Hind III sites of the expression vector pET-22b(+) / AP. The ligation product was transformed into competent Escherichia coli JM109 for transformation. The conversion method is as follows:

[0033] (1) Under sterile conditions, take 200 μL of competent cells and place them in a sterile microcentrifuge tube;

[0034] (2) Add 1-2 μL of recombinant plasmid to each tube, rotate gently to mix the contents, and place on ice for 30 minutes;

[0035] (3) Heat shock at 42°C for 90s (accurate), do not shake the centrifuge tube;

[0036] (4) Quickly transfer the centrifuge tube to an ice bath to cool the cells for 1-2 minutes;

[0037] (5) Add 800 μL of ordinary LB culture medium without antibiotics to each tube;

[0038] (6) Use a sterile spreader to spread 200 μL of the bacterial solution on an agar plate containing ampicillin, and place it flat...

Embodiment 3

[0040] Example 3: Construction of recombinant enzyme expression strains fused with parental short peptides

[0041] Competent Escherichia coli BL21 (DE3) was transformed. The conversion method is as follows:

[0042] (1) Under sterile conditions, take 200 μL of competent cells and place them in a sterile microcentrifuge tube;

[0043] (2) Add 1-2 μL of recombinant plasmid to each tube, rotate gently to mix the contents, and place on ice for 30 minutes;

[0044] (3) Heat shock at 42°C for 90s (accurate), do not shake the centrifuge tube;

[0045] (4) Quickly transfer the centrifuge tube to an ice bath to cool the cells for 1-2 minutes;

[0046] (5) Add 800 μL of ordinary LB culture medium without antibiotics to each tube;

[0047] (6) Use a sterile spreader to spread 200 μL of the bacterial solution on an agar plate containing ampicillin, and place it flat at 37°C for 20 minutes until the liquid is absorbed, then culture it upside down overnight, and observe.

[0048] Sele...

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Abstract

The invention discloses an enzymatic thermal stability improving method. Fusion expression of an amphiphilic short peptide and recombinase is carried out to obtain thermal stability-improved recombinase. The optimal sequence of the amphiphilic short peptide is DWLKAFYDKVAEKLKEAFKVQPYLDDWLKAFYDKVAEKLKEAF. The method has the advantages of substantial effect, simple process and convenient popularization, and provides a new method and idea for the rapid improvement of the thermal stability of industrial enzymes.

Description

technical field [0001] The invention relates to a method for improving the thermal stability of an enzyme, in particular to a method for improving the thermal stability of a recombined enzyme by using the fusion expression of a parental short peptide. Background technique [0002] Amphiphilic short peptides are small molecular peptides with hydrophilic and lipophilic abilities, which widely exist in the structures of membrane proteins and enzymes related to fat metabolism. The characteristics of its parents can help enzyme molecules combine with hydrophobic substrates and realize the positioning of enzyme molecules. [0003] Bioactive enzymes are widely used in industrial production, and improving the thermostability of enzymes is one of the research focuses of industrial enzymes. At present, the methods for improving the thermostability of enzymes mainly include: 1. Directed evolution: screening thermostable mutant strains through techniques such as site-directed mutation,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N9/26C12N9/10C12N15/70
CPCC11D3/386C12N9/54
Inventor 陈坚堵国成陆信曜刘松张娟
Owner JIANGNAN UNIV
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