Enzyme compound and self-assembled catalytic nanowire

A complex and self-assembly technology, applied in the direction of enzyme stabilization, can solve the problems of high cost, complex preparation, limited catalytic properties and stability, and achieve the effect of realizing length and control.

Inactive Publication Date: 2018-05-08
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention provides fibroblast protein in order to overcome the shortcomings in the prior art, such as limited enhancement of enzyme catalytic properties and stability, complex preparation, high cost, and the need to find suitable reagents, materials or methods for the specific enzyme. Use in Enhancing Enzyme Catalytic Activity and / or Enzyme Stability

Method used

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  • Enzyme compound and self-assembled catalytic nanowire
  • Enzyme compound and self-assembled catalytic nanowire
  • Enzyme compound and self-assembled catalytic nanowire

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] The preparation of embodiment 1Sup35-MPH enzyme complex

[0077] 1. Cloning of Sup35-MPH enzyme complex

[0078] Yeast prion protein self-assembly domain (the 1-61st amino acid of Sup35, referred to as Sup35, the specific sequence is shown in SEQ ID NO.1) and methyl parathion hydrolase (the 36th-331th amino acid of MPH) were combined by molecular cloning amino acids, the specific sequence shown in SEQ ID NO.2) is fused and connected through a flexible linker peptide (see the specific sequence shown in SEQ ID NO.3) to form a fusion protein Sup35-MPH, and then a His tag is fused at its N-terminus. Methyl parathion hydrolase (MPH) alone was used as a control. Specific steps are as follows:

[0079] 1) Using the plasmid pET28a as a template, using the primer MPH-Primer-1 to carry out PCR amplification to obtain the plasmid pET28a containing the sequence of the primer MPH-Primer-1;

[0080] 2) using the MPH gene fragment as a template, using the primer MPH-Primer-3 to per...

Embodiment 2

[0090] Example 2 Detection of Sup35-MPH enzyme complexes and nanowires

[0091] SDS-PAGE electrophoresis is carried out to the MPH and Sup35-MPH that embodiment 1 makes, the gel that obtains is carried out Coomassie brilliant blue staining, and its result is as follows figure 2 shown. Depend on figure 2 It can be seen that between 30KDa and 40KDa, there are clear and obvious bands around 40KDa, which correspond to free methyl parathion hydrolase protein (MPH) and Sup35-MPH enzyme complex respectively. Electrophoresis showed no obvious bands of miscellaneous proteins, indicating that the purity and expression level of the methyl parathion hydrolase protein (MPH) and Sup35-MPH enzyme complex prepared in Example 1 were high.

[0092] The self-assembled catalytic nanowire Sup35-MPH prepared in Example 1 was detected by electron microscopy, and the results were as follows image 3 As shown in A. Depend on image 3 A shows that the concentration and length distribution of nan...

Embodiment 3

[0093] The preparation of embodiment 3 Sup35-ATA-117 enzyme complex

[0094] 1. Cloning of Sup35-ATA-117 enzyme complex

[0095] Yeast prion protein self-assembly domain (1-61 amino acids of Sup35, referred to as Sup35, specific sequence is shown in SEQ ID NO.1) and sitagliptin aminotransferase (ATA-117, specific sequence is shown in SEQ ID NO.1) by molecular cloning Shown in NO.4) is fused and connected through a flexible linker peptide (see SEQ ID NO.3 for the specific sequence) to form a fusion protein Sup35-ATA-117, and then a His tag is fused to its N-terminus. Sitagliptin aminotransferase (ATA-117) alone was used as a control. Specific steps are as follows:

[0096] 1) Using the plasmid pET28a as a template, using the primer ATA-Primer-1 to carry out PCR amplification to obtain the pET28a vector containing the sequence of the primer ATA-Primer-1;

[0097] 2) Use the plasmid PUC57-ATA-117 as a template, and use the primer ATA-Primer-2 to perform PCR amplification to ob...

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Abstract

The invention provides application of fibroblast protein in enhancement of enzyme catalytic activity and / or enzyme stability. Through self-assembly of the fibroblast protein structure, enzyme molecules are displayed on the surface of the fibroblast protein nanometer structure at high density and in an array so as to form a linear nanometer structure with catalytic ability. The nanometer structuresimulates the natural regionalization state of enzyme molecules inside cells, so that the catalytic activity and stability of enzyme are improved under the condition that the enzyme molecules do not change. For example, the Michaelis constant Km of the Sup35-MPH in a nanowire state is not greater than 1 / 5 of that of free MPH, the catalytic constant Kcat is doubled, the maximum rate Vmax is improved by 26.5 times, and the specific activity is improved by 4.8 times; compared with free enzyme ATA-117, in the same reaction time, the substrate conversion rate of the Sup35-ATA-117 in the nanowire state can be improved by about 30%, and the time that the maximum conversion rate is reached is shortened by more than four times.

Description

technical field [0001] The invention relates to enzyme catalysts, in particular to an enzyme complex and self-assembled catalytic nanowires. Background technique [0002] As a kind of biocatalyst, enzyme has been widely used in various fields of production and life. In recent decades, with the continuous technological breakthrough of enzyme engineering, it has been widely used in industry, agriculture, medicine and health, energy development and environmental engineering. In the process of research and application of enzyme catalysis, people always expect that the higher the activity and stability of the enzyme, the better, and it has good catalytic performance. How to improve the catalytic ability of enzyme proteins is one of the research hotspots in the academic circles, and it is also an urgent problem to be solved in industrial production practice. Various traditional methods to improve the catalytic ability of enzyme proteins mainly include chemical modification, mole...

Claims

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Application Information

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IPC IPC(8): C12N9/96
CPCC12N9/96
Inventor 门冬张先恩魏翠华
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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