Laccases are oxidative enzymes containing 4 conserved copper heteroatoms. Laccases catalyze cleavage of bonds in lignin using radical chemistry, yet their exact specificity for bonds (such as the b-O-4 or C-C) in lignin remains unknown and may vary with the diversity of laccases across fungi, plants and bacteria. Bond specificity may perhaps even vary for the same enzyme across different reaction conditions. Determining these differences has been difficult due to 1: heterologous expression of soluble, active laccases has proven difficult and 2: activity assays for measuring exact bond type cleavage events have not been available. Here we describe the successful heterologous expression of functional laccases in two strains of <i>Saccharomyces cerevisiae</i>, including one we genetically modified with CRISPR. The expressed laccases were assayed for their ability to catalyze cleavage of b-O-4 or C-C bonds in model substrates using nanostructure-initiator mass spectrometry (NIMS), which allows determination of the specificity/preference of each of these enzymes. We describe differences in the bond preferences of these enzymes as a function of both protein sequence differences and pH and temperature profiles, leading to a potential future screening platform for directed evolution of laccases and other ligninolytic enzymes such as peroxidases.

High throughput expression of laccases in <i>Saccharomyces cerevisiae</i> and characterization of their bond cleavage specificity using nanostructure-initiator mass spectrometry

ACS Fall 2021

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.

High throughput expression of laccases in Saccharomyces cerevisiae and characterization of their bond cleavage specificity using nanostructure-initiator mass spectrometry