Incorporation of a stabilizing Ca(2+)-binding loop into subtilisin BPN'

Area of Science:

• Biochemistry and Molecular Biology
• Enzyme Engineering and Protein Stability

Background:

• Subtilisin BPN' is prone to autoproteolysis at elevated temperatures.
• Autoproteolysis occurs at specific peptide bonds (Ala48-Ser49 and Ser163-Thr164) located in flexible surface loops.
• Improving protease stability is crucial for industrial and biotechnological applications.

Purpose of the Study:

• To enhance the thermal stability of subtilisin BPN' against autoproteolysis.
• To investigate the role of specific sequence regions in protease stability.
• To explore the transfer of stabilizing elements from thermophilic homologues.

Main Methods:

• Identification of autolysis sites using protein isolation and N-terminal sequencing.
• Protein engineering by replacing susceptible regions with sequences from thermitase.
• Characterization of chimeric enzyme properties: Ca2+ binding, catalytic activity (kcat, KM), and thermal stability.
• Assessing enzyme stability via residual activity measurements against a specific substrate (sAAPF-pna).

Main Results:

• Two primary autolysis sites were identified in subtilisin BPN' at Ala48-Ser49 and Ser163-Thr164.
• A chimeric subtilisin BPN' incorporating a thermitase Ca2+-binding segment showed moderate Ca2+ affinity.
• This Ca2+-binding mutant exhibited 10-fold greater stability against thermal inactivation at 60°C in the presence of CaCl2.
• Mutations near the second autolysis loop did not improve stability and reduced it in some cases.

Conclusions:

• Strategic replacement of susceptible regions with sequences from thermophilic proteases can enhance stability.
• The introduction of a Ca2+-binding site significantly improved the thermal stability of subtilisin BPN'.
• Not all mutations derived from thermophilic homologues confer increased stability to mesophilic proteases.