Method for redesigning enzyme based on isothermal compression coefficient disturbance, application and mutant screened by method

A technology of isothermal compression and redesign, applied in the field of computational chemistry, can solve the problem of lack of globality in high-fluctuation regions, and achieve the effect of reducing screening workload, saving time and computing resources, and improving stability and activity.

Active Publication Date: 2022-03-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the above technical problems, the present invention provides a method that can simultaneously improve the stability and activity of enzymes, and the method is applicable to a variety of enzyme systems, has universal applicability, and overcomes the problem of determining high fluctuation regions in the prior art Lack of global issues

Method used

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  • Method for redesigning enzyme based on isothermal compression coefficient disturbance, application and mutant screened by method
  • Method for redesigning enzyme based on isothermal compression coefficient disturbance, application and mutant screened by method
  • Method for redesigning enzyme based on isothermal compression coefficient disturbance, application and mutant screened by method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1T1

[0060] Example 1T1 lipase

[0061] 1 Screening of high fluctuating area based on isothermal compression coefficient

[0062]The present example is the initial model with the primary model (PDB ID: 2DSN) of the wild-type T1 lipase (T1 lipase from GeoBacillus Zalihae Strain T1, using GromACS (2019.06 version). Molecular kinetic simulation. Applying the AMBER99 force field when simulated, putting the protein into a cube box filled with water, and the protein distance cassette is a shortest 1.0 nm, and the water model is TIP4P, then 5 sodium ions are added to charge balance. The system is used to minimize the system to ensure normal structure, the atomic distance is appropriate, the geometric configuration is reasonable, and then the NVT balance of 400 ps is performed under the cycle boundary conditions, and the Berendsen temperature is coupled to 313K, using Parrinell-Rahman pressure Coupling gradually increases from the atmosphere to the expected high pressure (100 bar, 500 bar, 100...

Embodiment 2

[0087] Example 2 Protein Glutamine Enzyme

[0088] 1 Screening of high fluctuating area based on isothermal compression coefficient

[0089] This example is the initial model with the crystal structure (PDB ID: 2ZK9) of wild-type protein glutamine (Pgase) (PDB ID: 2ZK9), using GromACS (2019.06 version). Molecular kinetic simulation. Applying the AMBER99 force field when simulated, putting the protein into a cube box filled with water, and the edge of the protein distance is 1.0 nm, the water model is TIP4P, and 3 chloride ions are added to charge balance. Using the 50000 step to minimize the system, under cycle boundary conditions, the NVT balance of 400 ps is performed, and the Berendsen temperature is coupled to 318K, and the Parrinell-Rahman pressure coupling gradually increases from the atmosphere to the expected high pressure (100 bar, 500 bar, 1000 bar, 2000Bar, 4000Bar). The balance removal limit of the system is applied to the finished product simulation. During the entire...

Embodiment 3

[0112] Example 3 xylanase

[0113] 1 Screening of high fluctuating area based on isothermal compression coefficient

[0114] The present example is the initial model of the crystal structure (PDB ID: 2DFC) of the wild-type xylase (PDB ID: 2DFC), the Monte Carlo sample algorithm based on the random generated variable, using the sampling mean of the function to approximate the estimation target The expectation of functions. Put the initial constructure into the system, the initial structure is randomly disturbed by RotAmertrialMover (), and the Monte Carlo sample algorithm uses a random number that meets uniform distribution within the interval [0, 1]. The algorithm obtains a series of predictive conformations, and the energy average of these conformations serves as an estimate of the average energy of the protein, and all simulations are repeated five times.

[0115] The secondary structure of the protein in this example is calculated by DSSP, and the structural analysis and visual...

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Abstract

The invention relates to a method for redesigning enzyme based on isothermal compression coefficient disturbance, application and a mutant screened by the method, and belongs to the technical fields of computational chemistry, bioinformatics, genetic engineering and protein engineering. The method comprises the following steps: screening out a high-fluctuation region, far away from an active center, on the surface of protein through isothermal compression coefficient disturbance, carrying out virtual saturation mutation on amino acid on the screened high-fluctuation region, and screening out mutants of which the Gibbs free energy is reduced after mutation, so as to obtain redesigned enzyme; wherein the disturbance mode of the isothermal compression coefficient is pressurization. The method provided by the invention overcomes the problems of lack of globality in screening of high-fluctuation regions and incapability of simultaneously improving the stability and activity of the enzyme in the prior art, provides a widely applicable method for design optimization of the enzyme, and improves the application value of the enzyme in industry.

Description

Technical field [0001] The present invention relates to calculation of chemical, biological information, genetic engineering, and protein engineering techniques, and more particularly to a method, application, application, and mutant, based on an isothermal compression coefficient disturbance, and a mutant to screen. Background technique [0002] Naturally present enzymes are often stressed by high temperatures, organic solvents in practical industrial applications, resulting in low stability of the enzyme, so that the stability of enzymes has always been a hot spot for production and research. There is currently a large number of research reports on enzyme heat stability and active transformation. Such as Truongvan, N. Et al., By research and catalytic H307 residues in the same ring in the same ring, D308A Or Y309A mutants exhibit enhanced substrate affinity and catalytic rate by expanding the active site, however, all mutant ESTK enzymes exhibit a reduced thermal stability, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G16C20/00C12N9/80C12N9/24C12N9/20
CPCG16C20/00C12N9/20C12N9/80C12N9/248C12Y305/01043
Inventor 夏小乐郑楠龙梦飞高玲王颖妤
Owner JIANGNAN UNIV
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