Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• Bacillus licheniformis alpha-amylase (BLA) exhibits high thermostability despite its mesophilic origin.
• The structural basis for BLA's thermal properties remains incompletely understood.
• Previous studies have proposed specific amino acid residues contributing to BLA thermostability.

Purpose of the Study:

• To elucidate the role of specific amino acid residues in BLA thermostability using structure-based mutagenesis.
• To identify residues critical for thermal stability and explore substitutions for enhanced enzyme performance.
• To pinpoint key structural determinants of BLA's heat resistance.

Main Methods:

• Site-directed mutagenesis was employed to create 175 BLA variants by replacing targeted residues.
• Mutant enzymes were analyzed in vitro for activity and thermostability via incubation at high temperatures.
• Residual activity measurements were used to quantify the impact of mutations on enzyme stability.

Main Results:

• Seven residues (Asp121, Asn126, Asp164, Asn192, Asp200, Asp204, Ala269) were intolerant to substitution, with some mutations causing significant instability.
• Substitution of asparagine residues at positions 172, 188, and 190 led to increased thermostability.
• Replacing Asn190 with phenylalanine resulted in a sixfold increase in half-life at 80°C, highlighting a key stabilization strategy.

Conclusions:

• BLA's thermostability determinants are concentrated in domain B and its interface with domain A, particularly around a triadic metal-binding site.
• A loop region (residues 178-199) is crucial for enzyme structural integrity, showing sensitivity to calcium removal.
• BLA's natural thermostability is not fully optimized and can be further improved by targeted mutations and removal of deamidation-prone residues.