Subtilisin BPN' variants: increased hydrolytic activity on surface-bound substrates via decreased surface activity

Area of Science:

• Biochemistry
• Enzyme Engineering
• Surface Chemistry

Background:

• Subtilisin BPN' (Bacillus amyloliquefaciens) is a protease with industrial applications.
• Enzyme adsorption to surfaces influences catalytic activity.
• Understanding enzyme-surface interactions is crucial for optimizing biocatalysis.

Purpose of the Study:

• To engineer subtilisin BPN' variants with altered surface adsorption properties.
• To investigate the relationship between enzyme adsorption and hydrolysis rate of surface-bound substrates.
• To identify amino acid substitutions affecting enzyme-surface interactions.

Main Methods:

• Site-directed and random mutagenesis were employed to create subtilisin BPN' variants.
• Protease adsorption and peptide hydrolysis rates were measured using surface-bound peptide substrates.
• Surface charge and hydrophobicity modifications were analyzed through amino acid substitutions.

Main Results:

• Most variants exhibited similar adsorption to native subtilisin BPN', but some showed increased or decreased adsorption.
• Contrary to hypothesis, variants with higher surface adsorption hydrolyzed surface-bound substrates slower.
• Variants with lower surface adsorption demonstrated faster hydrolysis rates.
• Amino acid substitutions altering surface charge/hydrophobicity, especially near the active site, significantly impacted adsorption and hydrolysis.

Conclusions:

• Enzyme adsorption level is inversely correlated with hydrolysis rate for surface-bound substrates.
• Weakly adsorbed enzymes may exhibit higher catalytic efficiency due to increased surface mobility.
• Modifying surface properties of enzymes offers a strategy to tune their performance in surface-bound reactions.