Engineering thermostability in subtilisin BPN' by in vitro mutagenesis

Area of Science:

• Biochemistry
• Protein Engineering
• Enzyme Kinetics

Background:

• Subtilisin, a bacterial serine protease, is widely used but can be limited by thermal instability.
• Enhancing enzyme thermostability is crucial for industrial applications and understanding protein structure-function relationships.

Purpose of the Study:

• To develop a method for isolating and identifying subtilisin mutants with enhanced thermostability.
• To characterize stabilizing mutations and engineer highly stable subtilisin variants.

Main Methods:

• Random mutagenesis of the cloned subtilisin BPN' gene using chemical mutagens.
• Selection of thermostable mutants via a plate assay screening for esterase activity at elevated temperatures.
• Oligonucleotide-directed mutagenesis to combine identified stabilizing mutations.

Main Results:

• Identified and characterized eight distinct mutations that enhance subtilisin thermostability.
• Constructed subtilisin variants by combining these mutations.
• Achieved a 12-fold increase in thermostability in a multi-mutant variant compared to wild-type subtilisin.
• Demonstrated that stabilizing mutations cause minor amino acid sequence alterations without radical changes in tertiary structure.

Conclusions:

• The developed procedure effectively identifies mutations that improve enzyme thermostability.
• Combining independent stabilizing mutations leads to a multiplicative increase in thermal stability.
• This work provides a foundation for predicting and introducing stabilizing changes in proteins.