Engineering of chimeric enzymes with expanded tolerance to ionic strength.

Area of Science:

• Biochemistry
• Microbiology
• Protein Engineering

Background:

• Antimicrobial resistance (AMR) is a critical global health crisis, leading to increased mortality from drug-resistant infections.
• The development of novel antibacterial agents is urgently needed to combat the rising threat of AMR.
• Peptidoglycan hydrolases are a promising class of bactericidal compounds with potential against resistant bacteria.

Purpose of the Study:

• To engineer chimeric enzymes by combining the EnpA catalytic domain (EnpA_CD) with SH3b cell wall-binding domains.
• To enhance the stability and activity of EnpA_CD under diverse environmental conditions, including serum.
• To explore the potential of protein engineering in developing new strategies against antimicrobial resistance.

Main Methods:

• Cloning and characterization of the M23 catalytic domain of EnpA from *Enterococcus faecalis*.
• Engineering of three chimeric enzymes by fusing EnpA_CD with distinct SH3b domains.
• Assessment of chimeric enzyme activity and tolerance to varying ionic strength, pH, and serum conditions.

Main Results:

• The engineered chimeric enzymes demonstrated improved tolerance to environmental conditions compared to the native EnpA_CD.
• Chimeric enzymes maintained sustained activity in both bovine and human serum.
• The addition of SH3b domains modulated the activity profile of the chimeric enzymes, broadening their applicability.

Conclusions:

• Fusion of SH3b-binding domains to EnpA_CD generates chimeras with enhanced tolerance to ionic strength and pH.
• Engineered enzymes remain active over a wider range of conditions, offering a straightforward method for developing antibacterial agents.
• Protein engineering of enzymes like EnpA_CD provides a viable strategy for creating tailored antibacterial agents to combat antimicrobial resistance effectively.