Hydrolysis of Milk Proteins by Immobilized Streptomyces griseus Pronase

Streptomyces griseus pronase was coupled covalently to porous succinamidopropyl-glass beads with 1-ethyl-3dimethylaminopropyl carbodiimide. The milk proteins, a-lactalbumin, ribonuclease A, bovine serum albumin,/3-1actoglobulin, whole casein, commercial casein, ~s~casein, 3-casein, and ~-casein were recirculated through a packed-bed column of immobilized pronase. The rate and extent of hydrolysis of the milk proteins were estimated by a 2,4,6-trinitrobenzenesulfonic acid assay which measured liberated a-amino groups. Rates of hydrolysis varied from .46 gtmol a-amino groups released min -1 100 mg glass beads -1 for ribonuclease A to 7.70/lmol a-amino groups released rain -I 100 mg glass beads -f for a commercial preparation of casein while the extent of hydrolysis ranged from 5% a-amino groups released in 30 min for ribonuclease A to 44% a-amino groups for asl-casein in 15 min. Sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis visualized the rapid loss of major casein bands and degradation of the generated peptide fragments. Pseudo-first order rate constant for N,N-dimethyl casein prepared by reductive alkylation was 5 10 -2 min -1 , while for casein, 7 10-2min -1 . Inclusion of 4 M urea with bovine serum albumin and stimultaneous exposure to