Insights into chitosan-cleaving enzymes by simultaneous analysis of polymers and oligomers via SEC-RI-ESI-MS

Abstract

Abstract The detailed characterization of chitosan-cleaving enzymes is of great importance to unveil structure-function relationships of this promising class of biomolecules, both for enzymatic fingerprinting analyses and to use the enzymes as biotechnological tools for the production of tailor-made chitosans for diverse applications. Analyzing polymeric substrates as well as oligomeric products has proven a suitable way to understand the enzymes’ actions but currently, this requires separate, rather laborious methods to obtain the full picture. Here, we describe size exclusion chromatography coupled to refractive index and electrospray ionization mass spectrometry detection (SEC-RI-ESI-MS) as a simple and fast way to semi-quantitatively analyze chitosan oligomers and to concomitantly determine the average molecular weight and its distribution for chitosan polymers. By sampling live from an ongoing enzymatic reaction, SEC-RI-ESI-MS offers the unique opportunity to analyze polymers and oligomers simultaneously – i.e. to follow the reduction in molecular weight of the polymeric substrate over the course of the digestion, while at the same time analyzing the emerging oligomeric products in a quantitative manner. In this way, a single simple analysis yields detailed insights into an enzyme’s action on a given substrate.