Protein engineering of Bacillus acidopullulyticus pullulanase for enhanced thermostability using in silico data...

Area of Science:

• Enzyme Engineering
• Biotechnology
• Protein Science

Background:

• Pullulanase enzyme activity is vital in starch processing, requiring high thermostability for industrial applications.
• Improving enzyme thermostability is crucial for maintaining catalytic efficiency at elevated temperatures.

Purpose of the Study:

• To engineer a more thermostable pullulanase using data-driven rational design methods.
• To identify key residues contributing to pullulanase thermostability through mutagenesis.

Main Methods:

• Employed four data-driven rational design methods: B-FITTER, proline theory, PoPMuSiC-2.1, and sequence consensus.
• Targeted 39 residues of Bacillus acidopullulyticus pullulanase for single and combined mutagenesis.
• Utilized fluorescence spectroscopy to analyze enzyme structural changes.

Main Results:

• The best mutant, E518I-S662R-Q706P, demonstrated an 11-fold increase in half-life at 60°C and a 9.5°C rise in melting temperature (Tm).
• The mutant's optimal temperature increased from 60°C to 65°C.
• Structural analysis indicated a more compact tertiary structure in the mutant, attributed to enhanced hydrophobicity, increased hydrogen bonds, and greater rigidity.

Conclusions:

• Combined data-driven rational design approaches are effective for enhancing industrial enzyme thermostability.
• The engineered pullulanase mutant shows significant potential for improved performance in high-temperature starch processing applications.