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A molecular modification design method for improving the catalytic efficiency of prolyl endopeptidase

A technology of prolyl endopeptidase and catalytic efficiency, which is used in the analysis of two-dimensional or three-dimensional molecular structure, sequence analysis, instruments, etc., can solve the problems of poor pH stability and inapplicability, and achieve the effect of improving catalytic activity

Active Publication Date: 2022-04-22
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few studies on the transformation of prolyl endopeptidases, and most prolyl endopeptidases are not suitable for food industry processing due to factors such as catalytic efficiency, poor temperature and pH stability

Method used

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  • A molecular modification design method for improving the catalytic efficiency of prolyl endopeptidase
  • A molecular modification design method for improving the catalytic efficiency of prolyl endopeptidase
  • A molecular modification design method for improving the catalytic efficiency of prolyl endopeptidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032]Example 1: Homology modeling and molecular docking of Sphaerobacter thermophiles prolyl endopeptidase

[0033] 1. Arrange and compare the enzymatic properties of prolyl endopeptidases of different species, select the prolyl endopeptidase derived from Sphaerobacter thermophiles as the object of transformation, the amino acid sequence of the prolyl endopeptidase As shown in SEQ ID NO.1, its nucleotide sequence is shown in SEQ ID NO.2.

[0034] 2. Search the amino acid sequence of the crystal structure of prolyl endopeptidase in the protein database (PDB database), and use the BLAST (local sequence alignment search basic tool) of the NCBI database to sequence the amino acid sequence of these prolyl endopeptidase Compare, the result is as figure 1 shown.

[0035] 3. Select the X-ray crystal structure (PDB ID: 2BKL) of Myxococcusxanthus prolyl endopeptidase (43.84% sequence identity) with the high similarity of Sphaerobacter thermophiles prolyl endopeptidase as template, us...

Embodiment 2

[0038] Example 2: Virtual Saturation Mutation of Residues Surrounding Prolyl Endopeptidase Substrates

[0039] Based on the stable enzyme-substrate complex structure obtained in Example 1, saturation mutations were performed around the substrate. Around P(Pro, proline) in the substrate sequence through the leap module of AMBER18 The 25 residues of prolyl endopeptidase in the range were subjected to virtual saturation mutation, and 575 virtual single-point mutants were generated. Virtual screening was carried out for all the above-mentioned virtual single-point mutants, and 9 with strong binding free energy were selected. a virtual single point mutant. The steps for virtual screening are as follows:

[0040] Constrained dynamics simulations were performed on all mutants to optimize their structures, even though the distance between the C atom of Pro in the substrate sequence and the O atom of Ser537 in the prolyl endopeptidase sequence was kept at The harmonic force consta...

Embodiment 3

[0054] Example 3: Molecular construction and expression of recombinant prolyl endopeptidase and site-directed mutants

[0055] The primer fragment names and sequences used in this embodiment are shown in the following table:

[0056]

[0057] Molecular Construction and Expression of Recombinant Prolyl Endopeptidase

[0058] 1. The Sphaerobacter thermophiles strain was cultured overnight at 37°C in 10 mL LB liquid medium. Use the bacterial genome DNA extraction kit to extract the genome of the Sphaerobacter thermophiles strain, use this as a template, and use P1 and P2 primers to obtain the prolyl endopeptidase target gene fragment (UniProtKB: D1C7Y4) by PCR amplification, the C-terminal of the target gene Containing His-tag, the pET-28a(+) plasmid was digested with EcoRI and NcoI restriction endonucleases respectively, and the recombinant DNA was obtained by reacting with T4 ligase in a metal bath at 16°C for 15 hours. Prolyl endopeptidase target gene PCR identification r...

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Abstract

The rational design method of the present invention to transform prolyl endopeptidase to improve its catalytic efficiency is mainly to obtain Sphaerobacter thermophiles prolyl endopeptidase through homology modeling, and obtain enzyme-substrate complex by molecular docking Structure, MD simulation stabilizes the structure, virtual saturation mutation of enzyme residues near the substrate, constrained MD simulation puts the enzyme in a perfect catalytic "near-offensive" conformation, uses MM / GBSA method to calculate binding free energy for virtual screening Mutant enzymes with improved potential catalytic efficiency were obtained. The enzyme was expressed by an Escherichia coli expression system, purified by a nickel column, and the enzyme activity and kinetic parameters of the recombinant enzyme for the nonapeptide substrate were determined by HPLC. The results show that the molecular transformation design method of the present invention can rapidly and effectively improve the catalytic activity of prolyl endopeptidase, which shows that the enzyme transformation method of the present invention is reliable.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a rational design method based on a catalytic perfect conformation developed by using a series of computer rational calculations based on the catalytic mechanism of prolyl endopeptidase, so as to improve the catalytic efficiency of the enzyme through directional transformation. Background technique [0002] Protein is one of the main nutrients and an important component in food, and its metabolites have many essential physiological activities in the human body. At present, exogenous proteases are usually added to proteins to obtain various types of bioactive peptides. On the premise of in-depth understanding of nutrition, health mechanisms, and precise, high-quality processing techniques, the method of adding proteases to functional foods has been obtained. Research extensively. However, there are still some limitations in the application of enzymes in industri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G16B5/00G16B15/30G16B30/10G16B50/30
CPCG16B5/00G16B15/30G16B30/10G16B50/30
Inventor 李晓琳韩艳芳张鲁嘉张增辉丛亚龙马明哲方波欢
Owner EAST CHINA NORMAL UNIV
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