Date

Monday 13 Jul 2026

Time

15:15 - 15:45

Location

BW008

Supervisor

Marcellus Ubbink

2nd reviewer

Anne Wentink

Jury

Anjali Pandit

β-Lactamases are amongst the most extensively studied enzyme families and are widely distributed across microbial life due to their central role in antimicrobial resistance. The oldest resurrected β-lactamase is the Precambrian last common ancestor of Gram-positive and Gram-negative bacteria (PNCA), estimated to be approximately three billion years old. PNCA is reconstructed through ancestral sequence reconstruction (ASR), a computational approach that infers ancestral amino acid sequences using phylogenetic modelling.

ASR is sensitive to model parameters, making it important to examine how reconstruction methods influence activity profiles. PNCA displays high thermal stability and measurable activity against multiple β-lactam substrates. These properties have been suggested to contribute to increased evolvability by enhancing tolerance for mutations while simultaneously providing latent activities that serve as starting points for the evolution of improved functions. However, it can be questioned to what degree thermostability and catalytic breadth are dependent on the reconstruction method of the ancestral enzyme. This thesis examines how reconstruction methods influence the functional activity profiles of PNCA variants.

Subsequently, this thesis also investigates whether PNCA can be further evolved through an error-prone PCR (epPCR) mutagenesis campaign to improve ampicillin hydrolysis activity. This approach aims to mirror the evolutionary trajectory of TEM-1, a modern β-lactamase that confers high resistance to ampicillin through efficient hydrolysis.

The results show that alternative reconstruction strategies introduce measurable variation in the individual kinetic parameters kcat and KM against nitrocefin, while catalytic efficiency (kcat/KM) and overall substrate profiles remain broadly conserved. The attempted epPCR campaign yielded no PNCA variant with improved ampicillin hydrolysis. However, initial mutational analysis indicates that PNCA tolerates sequence variation while maintaining activity, supporting its suitability as a starting point for studying evolutionary adaptation towards substrate specialisation.