Continuous enzymatic hydrolysis of beta-casein and isoelectric collection of some of the biologically active peptides in an electric field.
Among the milk proteins, bovine beta-casein has the peculiarity of containing in its sequence some peptides liable to interfere in mineral nutrition and some peptides with opioid (casomorphines), antihypertensive, and immunomodulatory activities. In this work we propose a novel type of multicompartment enzyme reactor, operating under an electric field, for the continuous hydrolysis of milk proteins such as beta-casein. The enzyme trypsin is trapped, with zwitterionic buffering ions and its substrate beta-casein, in solution between two isoelectric membranes having pI values encompassing the isoelectric point of the enzyme. Additionally, beta-casein is captured inside the same reaction chamber with the aid of sieving membranes, since its pI is too far away from the pI of trypsin. This setup permits the continuous operation at the pH of optimum of activity. The peptides, arising from tryptic hydrolysis of beta-casein, are removed under the influence of the electric field and collected in different chambers in which they are isoelectric and isoionic as well. The purity of the peptides collected is ascertained by capillary zone electrophoresis and their identity confirmed by N-terminal sequencing and MALDI-TOF mass spectrometry. This setup allows continuous harvesting of some biologically-active peptides in a pure form. The major advantages of such a reactor system over conventional batch reactors are the great increase in enzyme utilization efficiency and the overall reactor productivity.
Righetti
P G
PG
Department of Agricultural and Industrial Biotechnologies, University of Verona, Italy. RIGHETTI@IMIUCCA.CSI.UNIMI.IT
Nembri
F
F
Bossi
A
A
Mortarino
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D019075:Electrophoresis, Capillary; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D002364:Caseins; D010446:Peptide Fragments; D014357:Trypsin