Improve thermostability of Bacillus sp. TS chitosanase through structure-based alignment

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• Chitosanases are crucial enzymes for producing chitooligosaccharides, which have significant medical applications.
• Enzymatic synthesis offers a promising route for large-scale production, but enzyme stability is a key challenge.
• The chitosanase from Bacillus sp. TS exhibits moderate thermostability, limiting its industrial applicability.

Purpose of the Study:

• To enhance the thermostability of Bacillus sp. TS chitosanase through protein engineering.
• To identify specific mutations that improve enzyme half-life and catalytic efficiency at elevated temperatures.
• To investigate the structural basis for improved thermostability in engineered chitosanase variants.

Main Methods:

• Site-directed mutagenesis was employed to alter specific serine residues identified through structural comparison.
• Thermostability was assessed by measuring enzyme half-life at 60 °C.
• Enzyme kinetics, including K_m values, were determined to evaluate substrate binding and catalytic activity.
• 3D structures of mutants were analyzed to understand the mechanisms of enhanced stability.

Main Results:

• Three single mutants (S265G, S276A, S347G) exhibited significantly increased thermostability compared to the wild-type enzyme.
• The S265G mutant showed a half-life of 34.57 min, S276A of 36.79 min, and S347G of 7.2 min at 60 °C.
• Double and triple mutants demonstrated synergistic effects, resulting in even greater thermostability than single mutants.
• Mutations minimally impacted substrate binding affinity (K_m), while S265G showed reduced activity.

Conclusions:

• Targeted mutagenesis of specific serine residues is an effective strategy for enhancing chitosanase thermostability.
• Engineered chitosanases with improved thermal stability hold great potential for industrial-scale enzymatic synthesis of valuable chitooligosaccharides.
• Structural analysis provides insights into the molecular mechanisms underlying enzyme thermostabilization.