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Method for improving hydrolase robustness in combination with high-pressure molecular dynamics simulation and free energy calculation

A molecular dynamics, hydrolytic enzyme technique, applied in the field of computational chemistry

Pending Publication Date: 2021-03-30
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the above software can analyze the impact of mutations on protein molecules based on structure, they rely on static structures to evaluate the impact of mutations on protein structure and function

Method used

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  • Method for improving hydrolase robustness in combination with high-pressure molecular dynamics simulation and free energy calculation
  • Method for improving hydrolase robustness in combination with high-pressure molecular dynamics simulation and free energy calculation
  • Method for improving hydrolase robustness in combination with high-pressure molecular dynamics simulation and free energy calculation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: T1 lipase

[0054] 1 Screening of highly robust related regions and sites

[0055] In this example, the crystal structure (PDB id: 2dsn) of wild-type T1 lipase (T1 lipase from Geobacillus zalihae strain T1, T1 lipase) was used as the initial model, and the mutant was modeled using SWISS-MODEL. Gromacs (version 2019.03) performs molecular dynamics simulations.

[0056] The shortest distance from the edge of the box is 1.0nm, and the water model uses TIP4P, because this model is one of the most reliable water models for studying pressure effects, and then 5 Na + Neutralizing the charge brings the entire system into equilibrium. The 50000-step steepest descent method is used to minimize the energy of the system to ensure that the structure is normal, the distance between atoms is appropriate, and the geometric configuration is reasonable, and then a 400ps constant temperature and constant volume ensemble (NVT) balance is performed under periodic boundary conditi...

Embodiment 2

[0101] Embodiment 2: Rhizomucor miehei lipase (RML lipase)

[0102] 1 Screening of highly robust related regions and sites

[0103] The crystal structure of RML lipase (PDB id: 4tgl) was downloaded from the Protein Data Bank as the research object, and the downloaded crystal structure was corrected and optimized with the Repair and Optimize programs in FoldX suite 5.0. The MD simulation uses the GROMACS 2019.03 installation package. The AMBER99 force field is applied during the simulation. The protein is placed in a cubic box filled with water, and the distance between the protein and the edge of the box is at least 1.0nm. The water model uses TIP4P. Since this model is a research One of the most reliable water models for pressure effects, then add 10 sodium ions for charge balance. The steepest descent method with 50,000 steps is used to minimize the system to ensure normal structure, proper distance between atoms, and reasonable geometric configuration. Then, under periodic...

Embodiment 3

[0116] Example 3: Carbamate Hydrolase

[0117] 1 Screening of highly robust related regions and sites

[0118] The templates with high homology in the PDB database were used to model separately, and the Glutamyl-tRNA(Gln)amidotransferase subunit A (PDBID :2G5H) as a template, use the online three-dimensional structure simulation program SWISS-MODEL (http: / / swissmodel.expasy.org / interactive) to urethane hydrolase (EC 3.5.1.75, Urethanase, its amino acid sequence is shown in the sequence listing, Derived from Lysinibacillus fusiformis SC02) for three-dimensional structure modeling, under the conditions of 1Bar, 100Bar, 500Bar, 1000Bar, 2000Bar, 4000Bar pressure and 313K, molecular dynamics simulations were performed on UH, and the search for enzyme molecules Instability-associated structures or regions. Analysis of the B-factor value of each amino acid of the UH protein by FlexServ software shows that the B-factor response value of Leu327 in the sequence is the highest, indica...

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Abstract

The invention discloses a method for improving hydrolase robustness in combination with high-pressure molecular dynamics simulation and free energy calculation, and belongs to the fields of computational chemistry, bioinformatics, genetic engineering and protein engineering. According to the method, high-pressure disturbance is combined with simulation calculation of a molecular dynamics model tosearch for a strong-robustness related region and a key amino acid site, virtual saturation mutation is conducted on amino acid on the key region, and a mutant library is constructed; and then, multi-scale free energy calculation software is used for carrying out stability prediction on the mutants from protein dynamics, protein thermodynamics and prediction algorithms, and false positive mutantsare eliminated as much as possible through multiple rounds of virtual selection. By utilizing the method to construct and screen the hydrolase mutant, the frequency of the positive mutant is as high as 62.5%, the screening workload is effectively reduced, the obtained mutant is strong in robustness, and the method has very high industrial application potential.

Description

technical field [0001] The present invention relates to the fields of computational chemistry, bioinformatics, genetic engineering and protein engineering, relates to improving the robustness of hydrolytic enzymes by combining high-pressure molecular dynamics simulation and free energy calculation, and specifically relates to a screening method based on high-pressure molecular dynamics simulation combined with free energy calculation Methods to improve the robustness of hydrolases. Background technique [0002] Hydrolase (EC 3, Hydrolase) is a general term for a class of enzymes that catalyze hydrolysis reactions, including lipases, esterases, proteases, etc., which can catalyze various reactions, such as transesterification, esterification, and transesterification. Due to good catalytic performance, hydrolytic enzymes have been widely used in industrial fields such as food, medicine, oil production and processing, daily necessities and cosmetics. However, the actual indust...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G16C10/00G16B15/00C12N9/20C12N9/80
CPCG16C10/00G16B15/00C12N9/20C12N9/80C12Y301/01003C12Y305/01075G16B40/00G16B15/20G16B20/50G16B5/20
Inventor 夏小乐郑楠张泽华
Owner JIANGNAN UNIV
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