Liquid chromatography–mass spectrometry analysis of enzyme-hydrolysed carboxymethylcellulose for investigation of enzyme selectivity and substituent pattern

A series of celloendoglucanases: Bacillus agaradhaerens Cel 5a, Humicola insolens Cel 5a, H. insolens Cel 7b, H. insolens Cel 45a, Trichoderma reesei Cel 7b, and T. reesei Cel 45a were used to hydrolyse carboxymethylcellulose (CMC) and the hydrolysis products were investigated with a novel liquid chromatography–mass spectrometry (LC–MS) method. Separation was achieved using a graphitised carbon chromatographic column which allowed the use of electrospay compatible eluents. Analysis of the compounds produced during enzyme hydrolysis of CMC is used to understand enzyme selectivities and substitution pattern of CMC. Conventional high-performance anion-exchange chromatography (HPAEC)–pulsed amperometric detection (PAD), size-exclusion chromatography (SEC)–refractive index (RI) detection, and reducing end analysis are also used to analyse enzyme-hydrolysed CMC. The LC–MS method presented allows for a more detailed investigation of hydrolysis products, which facilitates characterisation of both enzymes and substrates.