Coupling trypsin enzyme onto chromatographic supports provides a platform to reuse the enzyme and automate the hydrolysis process. A monolithic chromatographic support, such as Convective Interaction Media (CIM®), enables mass transfer of molecules within its channels exclusively by convective flow. This results in enzymatic conversion which is not limited by diffusion, making CIM® monoliths ideal for the preparation of immobilised monolith enzymatic reactors (IMERs). BIA Separations offers CIMac™ trypsin IMER with a bed volume of 0.1 mL as analytical platform for mass spectrometry (MS)-based proteomics. Larger volume IMERs (up to 80 mL) are available for industrial scale production of protein hydrolysates. The following example describes the enzymatic production of β-Lactoglobulin (β-Lg) hydrolysates using monoliths with 2 μm (N1) or 6 μm (N2) average channel diameter.
A supernatant from Phanerochaete chrysosporium cultivation was loaded on CIM® QA Disk, and elution was effected by a linear gradient at a flow rate of 3 mL/min (9 CV/min). Baseline separation of isoenzymes H2, H6/H7, H8 and H10 was achieved in less than 3 minutes.
50 mL of crude enzyme solution were loaded on CIM® DEAE 8 mL tube and eluted by a linear gradient at a flow rate of 1.2 mL/min. Analysis of the elution fraction resulted in 86.1% recovery and 4 times concentration of the sample, having a purity of >95%.