Hydrolysis of β-lactoglobulin by four different proteinases monitored by capillary electrophoresis and high performance liquid chromatography

The hydrolysis of beta-lactoglobulin (beta-lg) by four enzymes [porcine trypsin (PT), Fusarium oxysporum trypsin (FOT), Bacillus licheniformis proteinase (BLP) and Bacillus subtilis proteinase (Neutrase(R))] was characterised by size exclusion-and reversed phase high performance liquid chromatography (SE-HPLC and RP-HPLC) and by capillary electrophoresis (CE). After 24 h of hydrolysis, all beta-lg had been degraded by PT and BLP, but a large part of the protein was still intact after hydrolysis by FOT or Neutrase(R). The main fraction of peptides was found to have MWs ranging from 1.0 to 3.0 kDa. The hydrolysis catalysed by each enzyme resulted in different peptide profiles by RP-HPLC and CE. Hydrolysates produced by PT or FOT are resolved into 18 peaks, while BLP hydrolysates were resolved into 25 peaks, corresponding well to the numbers of possible cleavage sites in beta-lg. Neutrase(R), with a broad specificity, produced the largest number of peptides. Seven peptides from PT hydrolysis, eight from FOT hydrolysis and one from BLP hydrolysis were identified by mass spectrometry (MS) and Edman degradation. (C) 1997 Elsevier Science Ltd. All rights reserved.