Functional expression of horseradish peroxidase in E. coli by directed evolution

Area of Science:

• Biotechnology
• Enzyme Engineering
• Molecular Biology

Background:

• Developing bacterial expression systems for enzymes like horseradish peroxidase (HRP) is crucial for industrial applications.
• Previous attempts to express wild-type HRP in Escherichia coli yielded minimal detectable activity.
• Cytochrome c peroxidase (CcP) expression served as a positive control, showing high activity in the supernatant.

Purpose of the Study:

• To establish a bacterial expression system for horseradish peroxidase (HRP).
• To improve HRP expression and activity using directed evolution.
• To identify mutations that enhance protein folding and stability in E. coli.

Main Methods:

• HRP gene was cloned into the pET-22b(+) vector, fused to the PelB signal peptide.
• Directed evolution involved random mutagenesis and gene recombination, followed by screening in a 96-well microplate format.
• Screening identified clones with enhanced HRP activity and facilitated further evolution for thermostability and H2O2 resistance.

Main Results:

• Wild-type HRP expression showed no detectable activity with isopropyl-beta-D-thiogalactopyranoside (IPTG), but weak activity was observed without IPTG.
• The first generation of directed evolution yielded a clone with 14-fold higher HRP activity (approx. 110 microg/L).
• This expression level enabled subsequent directed evolution for improved enzyme properties.

Conclusions:

• Directed evolution is effective in identifying mutations that improve protein folding efficiency in E. coli.
• This approach facilitates the development of bacterial expression systems for enzymes lacking such systems.
• The strategy is valuable for optimizing industrial biocatalysts, including HRP.