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Method for improving thermal stability of bacillus subtilis lipase A

A Bacillus subtilis, thermal stability technology, applied in the field of enzyme engineering, can solve the problems of complex process, limit the work progress in the field of protein transformation, large screening capacity, etc., achieve improved thermal stability, avoid large screening capacity, and simplify rational design The effect of the method

Active Publication Date: 2014-08-06
NANJING UNIV OF TECH
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Problems solved by technology

Although there have been a large number of reports on the modification of enzyme thermal stability, the traditional rational design method is limited by the complexity of protein structure and function relationship, while the irrational design method needs to face difficulties such as large screening capacity and complicated process. The shortcomings of these two methods have limited the progress of work in the field of protein engineering to some extent.

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

Examples

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Embodiment 1

[0024] This example illustrates the method of obtaining the crystal structure of Bacillus subtilis lipase A in step 1) of the present invention and analyzing the loop region with higher flexibility in it by means of molecular dynamics. Bacillus subtilis lipase A ( Bacillus subtilis LipA, PDB: 1I6W) was the research target, and the crystal structure of Bacillus subtilis lipase A was obtained by searching the RCSB database; the obtained Bacillus subtilis lipase A was subjected to 20 ns molecular dynamics simulation ( figure 2 ), and extract the equilibrium time period to analyze the root mean square fluctuation RMSF of Bacillus subtilis lipase A ( image 3 ).

Embodiment 2

[0026] This example illustrates the method of determining the glycine (Gly) residue in the highly flexible Loop region as the mutation site by proline-binding effect analysis in step 2) of the present invention. According to the RMSF diagram of Bacillus subtilis lipase A obtained in step 1), the Pymol visualization software combined with the "proline effect" theory analysis, finally, the results of the mutation sites obtained in this round of screening are: Gly153, Gly155, Gly158, Gly111, Gly116, Gly46, Gly52.

Embodiment 3

[0028] This example illustrates the method of step 3) through the molecular dynamics simulation analysis, the method of screening the disabling mutation of Gly to Pro on the thermostability of Bacillus subtilis lipase A.

[0029] The Gly153Pro, Gly155Pro, Gly158Pro, Gly111Pro, Gly116Pro, Gly46Pro, and Gly52Pro mutants were constructed using the online server SWISS-Model, and the constructed mutant models were evaluated and optimized using Verify_3D. The kinetic simulation of Bacillus subtilis lipase A and its mutants was carried out using the GROMACS4.5.4 software package. The simulation steps mainly include the following steps:

[0030] The first step uses the pdb2gmx command to add missing hydrogen atoms in the protein. And use the cube box to fill the SPC solvent water model and the GROMOS9653a6 force field. The command is:

[0031] pdb2gmx -f LipA.pdb -o.gro -p LipA.top -i .itp -water spc -ignh

[0032] editconf -bt cubic -f LipA.gro -o LipA.gro -d 0.9

[0033] The se...

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Abstract

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. According to the method, bacillus subtilis lipase A is taken as a study object, a crystal structure of bacillus subtilis lipase A is obtained from the RCSB (Research Collaboratory for Structural Bioinformatics) database firstly, and flexibility of different Loops of bacillus subtilis lipase A is analyzed by molecular dynamics; according to the rational analysis and combined with a proline effect theory, glycine residues which are located in the Loops with higher flexibility and obtained through screening are mutated to proline; a mutation result obtained through screening is verified through molecular dynamics simulation; and finally, hotspot residues with modified thermal stability are verified through a thermal stability experiment of a bacillus subtilis lipase A mutant strain.

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] Lipase, namely triacylglycerol acyl hydrolase, is a special ester bond hydrolase, which can catalyze the hydrolysis reaction of oil at the oil-water interface to generate fatty acid and glycerol, monoglyceride or diester. Lipase A secreted by Bacillus subtilis is a kind of biocatalyst with good application prospect in food, medicine, chemical industry and other fields. Bacillus subtilis lipase is minimal α / β folded lipase, and lacks the α The "lid structure" formed by the helix, so that there is no interface activation effect that other lipases have. However, Bacillus subtilis lipase A is extremely sensitive to temperature, and its enzyme activity will drop sharply when the temperature exceeds 40°C. [0003] Thermostability of enzymes is one of the important at...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12R1/125
CPCC12N9/20C12N9/96C12Y301/01003
Inventor 黄和江凌李晓彤李霜胡燚
Owner NANJING UNIV OF TECH
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