Engineering of a CPC acylase using a facile pH indicator assay

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Industrial Microbiology

Background:

• Cephalosporin C (CPC) acylase is crucial for synthesizing 7-aminocephalosporanic acid (7-ACA), a vital intermediate for cephalosporin antibiotics.
• Current CPC acylase applications are limited by low activity, substrate, and product inhibition.

Purpose of the Study:

• To engineer Pseudomonas SE83 CPC acylase (acyII) to overcome limitations in 7-ACA production.
• To reduce substrate and product inhibition for improved enzyme efficiency.

Main Methods:

• Two rounds of combinatorial active-site saturation testing (CASTing) were used to obtain a substrate inhibition-free mutant.
• A novel pH indicator assay was developed for real-time monitoring of product inhibition.
• Site-directed saturation mutagenesis libraries were screened using the developed assay.

Main Results:

• A mutant H57βA/H70βY was generated, eliminating substrate inhibition.
• A triple mutant H57βA/H70βY/I176βN was identified, showing a 3.26-fold increase in catalytic rate (kcat) and a 3.08-fold increase in product inhibition constant (KIP) compared to the wild type.
• The developed pH indicator assay proved effective for screening enzyme variants.

Conclusions:

• The engineered CPC acylase variants, particularly the triple mutant, demonstrate reduced product inhibition and enhanced activity.
• The novel pH indicator assay is a valuable tool for further enzyme optimization.
• These advancements hold significant potential for the industrial production of 7-ACA and cephalosporin antibiotics.