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,

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

Example Embodiment

[0076] Example 1 Preparation of Sup35-MPH enzyme complex

[0077] 1. Sup35-MPH enzyme complex clone

[0078] Through molecular cloning, the yeast prion protein self-assembly domain (amino acids 1-61 of Sup35, referred to as Sup35, see SEQ ID NO. 1 for the specific sequence) and methyl parathion hydrolase (36-331 of MPH) Two amino acids, see SEQ ID NO. 2 for the specific sequence) fusion-linked through a flexible connecting peptide (see SEQ ID NO. 3 for the specific sequence) to form the fusion protein Sup35-MPH, and then a His tag is fused to its N-terminus. A single methyl parathion hydrolase (MPH) was used as a control. Specific steps are as follows:

[0079] 1) Using plasmid pET28a as a template, PCR amplification was performed with primer MPH-Primer-1 to obtain plasmid pET28a containing primer MPH-Primer-1 sequence;

[0080] 2) Using the MPH gene fragment as a template, the primer MPH-Primer-3 is used for PCR amplification to obtain the MPH gene fragment with His-tag at the N en...

Example Embodiment

[0090] Example 2 Detection of Sup35-MPH enzyme complex and nanowire

[0091] The MPH and Sup35-MPH prepared in Example 1 were subjected to SDS-PAGE electrophoresis, and the resulting gel was stained with Coomassie brilliant blue. The results are as follows figure 2 Shown. by figure 2 It can be seen that between 30KDa and 40KDa, there are clear and obvious bands near 40KDa, which respectively correspond to free methyl parathion hydrolase protein (MPH) and Sup35-MPH enzyme complex. The electrophoresis showed no obvious contaminated protein bands, indicating that the methyl parathion hydrolase protein (MPH) and Sup35-MPH enzyme complex prepared in Example 1 had high purity and expression.

[0092] The self-assembled catalytic nanowire Sup35-MPH prepared in Example 1 was detected by electron microscope, and the results are as follows image 3 Shown in A. by image 3 A shows that the concentration and length distribution of the nanowires formed by self-assembly are uniform.

Example Embodiment

[0093] Example 3 Preparation of Sup35-ATA-117 enzyme complex

[0094] 1. Sup35-ATA-117 enzyme complex clone

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

[0096] 1) Using plasmid pET28a as a template, PCR amplification was performed with primer ATA-Primer-1 to obtain pET28a vector containing primer ATA-Primer-1 sequence;

[0097] 2) Using the plasmid PUC57-ATA-117 as the template, the primer ATA-Primer-2 was used for PCR amplification to obtain the His-tag at the N end tag -ATA-117 gene fragment;

[0098] 3) Using plasmid pET28a...

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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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