High-Throughput Screening Using UHPLC-MS To Characterize the Subsite Specificities of Chitosanases or Chitinases

Partially acetylated chitosan oligosaccharides (paCOS), consisting of β-1,4-linked <i>N</i>-acetyl-d-glucosamine and d-glucosamine units, possess diverse bioactivities that can be used for applications in, e.g., biomedicine, agriculture, and pharmaceutics. Establishing structure-function relationships and revealing modes of action requires the availability of structurally defined paCOS that can best be produced using chitin- and chitosan-modifying enzymes, such as chitinases and chitosanases, with known and defined subsite specificities. To enlarge the spectrum of such enzymes and, consequently, defined paCOS available, we have developed a two-step, microtiter plate-based high-throughput screening assay that allows quantification of the activity and subsite specificities of chitosan hydrolases. In a first step, the activities of the enzymes are quantified using a reducing end assay, and enzymes with sufficient activity are then screened for their subsite specificities using mass spectrometric analysis of their products when acting on well-defined chitosan polymers as substrates. The rapid UHPLC-ELSD-ESI-MS² method does not require labeling steps or addition of standards, and the principal component analysis of the fragment ion intensities of just two isomeric oligomer groups, GlcNAc₁GlcN₃ and GlcNAc₂GlcN₂, sufficed to identify, in a directed evolution, the site-saturation mutagenesis library of <i>Bacillus</i> sp. MN chitosanase consisting of 167 muteins, enzymes that significantly differed in their subsite specificities from the wildtype enzyme. Detailed analyses of a few selected muteins proved that the screening method is efficient and accurate in predicting altered subsite specificities.