Method for improving heat stability of lipase A of bacillus subtilis

A Bacillus subtilis, thermostable technology, applied in the field of enzyme engineering, can solve the problems of complex process, high price, large screening workload of high-throughput screening methods, etc.

Active Publication Date: 2013-08-14
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there have been a large number of reports on the modification of enzyme thermostability, most of the experimental programs focus on directed evolution high-throughput screening and high-performance computer screening.
Although the former does not need to understand the relationship between protein structure and function, the high-throughput screening method is often accompanied by a large screening workload and complex process; the latter has strict requirements on computer computing power and often requires the support of expensive high-performance servers

Method used

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  • Method for improving heat stability of lipase A of bacillus subtilis
  • Method for improving heat stability of lipase A of bacillus subtilis
  • Method for improving heat stability of lipase A of bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] This example illustrates the method of step 1) of the present invention for predicting hot spots of amino acid mutations in Bacillus subtilis lipase A.

[0025] Taking Bacillus subtilis lipase A (Bacillus subtilis lipase A, PDB: 1I6W) as the research target, using the HotSpot Wizard online server to virtually screen mutation hotspots, and statically analyze the primary sequences of more than 50 Bacillus subtilis lipase family proteins By comparison, the mutability rate and mutation results of each residue of Bacillus subtilis lipase A are obtained, the amino acid residue sites with high mutation rate are screened, and functional amino acids around the catalytic active center and substrate channel are excluded, thereby predicting amino acid mutations hotspot area.

[0026] This round of screening for thermally stable points was performed on residues with a mutation rate of 9. For example, in the first round, residues with a mutation rate of 9 cannot be screened for reas...

Embodiment 2

[0029]This example illustrates step 2) of the present invention, a method for determining the amino acid sites located on the surface of the protein and in a relatively flexible region.

[0030] Use Pymol visualization software and B-factor (B-factor) to determine the amino acid positions on the surface of the protein and in the more flexible region.

[0031] Use PyMOl to visualize amino acid processes located on protein surfaces:

[0032] PyMOL>load 1I6W.pdb (open the three-dimensional structure of Bacillus subtilis lipase A);

[0033] PyMOL>show surface (protein is expressed in surface form);

[0034] PyMOL>color white (the protein surface is displayed in white);

[0035] PyMOL>select3, resi3 (3 represents the third residue, and the following residues are displayed in this way);

[0036] PyMOL>color red,3 (show the third amino acid in red to determine whether it is on the surface of the protein);

[0037] PyMOL>color white,3 (make the third amino acid epitope white).

...

Embodiment 3

[0042] This example illustrates the method of combining the results obtained in Example 1 and Example 2 to analyze and determine amino acid mutation sites related to thermal stability.

[0043] Combine the results obtained in Examples 1 and 2 to determine the amino acid mutation site related to thermostability, if the mutation becomes aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg ) Any one of the four amino acid residues can improve the thermostability of the enzyme.

[0044] Using Pymol visualization software, with this amino acid as the center, in 4 The process of searching for the oppositely charged amino acid residues in the above four amino acids within the scope.

[0045] PyMOL>select60, resi60 (select amino acids that can be mutated);

[0046] PyMOL>select near60,60expand4 (select 4 range of residues, determine if it is the oppositely charged residue).

[0047] Through the above analysis, the following high mutation rate residues and result...

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Abstract

The invention belongs to the technical field of enzyme engineering, and relates to a method for improving the heat stability of lipase A of bacillus subtilis. According to the method, the lipase A of the bacillus subtilis is used as an object of study, and the method comprises the following steps of: firstly, screening mutational hot spot areas; secondly, determining the amino acid site of an area which is positioned on the surface of protein and is higher in flexibility; thirdly, carrying out rational analysis by combining the two steps, mutating the screened amino acid site into any one of four amino acid residues including aspartic acid, glutamic acid, lysine and arginine, taking the amino acid as a center, and searching the amino acid residues with opposite charges in the four amino acids in a range of 4 angstroms to determine a mutation site and a mutation residue; fourthly, on the basis of the acknowledge of an enzyme structure and a functional relationship, resolving ionic bonds, and introducing a mechanism of action for stabilizing the protein structure of the lipase A of the bacillus subtilis; and finally, proving hot spot residues with modified heat stability through hot stability experiments of mutant strains of the lipase A of the bacillus subtilis.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering and relates to a method for improving the thermal stability of bacillus subtilis lipase A. Background technique [0002] Enzymes are a very important class of proteins that can catalyze a variety of chemical reactions under mild conditions in living organisms. At present, lipase is widely used in industrial production as an important class of biocatalysts. Bacillus subtilis lipase A is the smallest lipase (19 kDa) and lacks the lid structure typical of most lipases and thus has no "interface effect". Bacillus subtilis lipase A belongs to the "true esterase" subfamily I.4, and has good stability under neutral or slightly alkaline conditions. However, subfamily I.4 lipases are extremely sensitive to temperature. The most suitable temperature for Bacillus subtilis lipase A is 35°C, and its enzyme activity drops sharply when the temperature exceeds 40°C. [0003] Thermal stability is an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12R1/125
Inventor 黄和张洋江凌胡燚李晓彤张红漫
Owner NANJING UNIV OF TECH
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