Engineering CotA Laccase for Acidic pH Stability Using Bacillus subtilis Spore Display.

Area of Science:

• Biotechnology
• Enzyme Engineering
• Protein Engineering

Background:

• Traditional protein display methods face challenges with protein folding and cell viability.
• Bacillus subtilis spores offer a stable and viable platform for protein immobilization, maintaining genotype-phenotype linkage.
• Spore-based display leverages natural sporulation and chaperone systems to facilitate correct protein folding.

Purpose of the Study:

• To engineer CotA as a whole-cell biocatalyst with improved pH stability using Bacillus subtilis spore display.
• To enhance the operational stability and reusability of the CotA biocatalyst for industrial applications.

Main Methods:

• Screening of a library of approximately 3,000 Bacillus subtilis clones displaying CotA variants.
• Directed evolution and site-directed mutagenesis to identify and combine beneficial mutations for improved pH stability.
• Characterization of wild-type (wt) CotA and engineered variants (E498G, T480A/E498G) for activity, stability, and reusability.

Main Results:

• A single mutation (E498G) significantly increased the half-life of CotA at pH 4 by 24.8-fold compared to wt-CotA.
• A double mutant (T480A/E498G) exhibited a 62.1-fold greater half-life at pH 4 than wt-CotA.
• Engineered CotA variants (E498G and T480A/E498G) demonstrated 3.7- and 5.3-fold higher product yields, respectively, after seven recycling cycles over 42 hours.

Conclusions:

• Bacillus subtilis spore display is an effective strategy for enhancing enzyme pH stability and biocatalytic performance.
• Engineered CotA variants exhibit superior operational stability and reusability, making them promising for industrial biocatalysis.
• This approach overcomes limitations of traditional protein display, offering a robust platform for enzyme engineering.