Cross-linked enzyme aggregates (CLEAs) of PepX and PepN – production, partial characterization and application of combi-CLEAs for milk protein hydrolysis

Abstract The proline-specific X-prolyl-dipeptidyl aminopeptidase (PepX; EC 3.4.14.11) and the general aminopeptidase N (PepN; EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 were individually immobilized as cross-linked enzyme aggregates (CLEA). The influence of the precipitant, precipitant concentration, cross-linking time, cross-linking agent concentration, the proteic feeder casein, and sodium borohydride was investigated to find the optimal conditions for CLEA preparation. Ammonium sulfate was selected as the most efficient precipitation agent for both enzymes. After optimization of the CLEA preparation conditions (PepX: 3 M (NH 4 ) 2 SO 4 , 2 h cross-linking time, 100 mM glutaraldehyde, 0.087 mg/mL casein, no addition of sodium borohydride; PepN: 4 M (NH 4 ) 2 SO 4 , 4 h cross-linking time, 50 mM glutaraldehyde, no addition of casein and sodium borohydride), the highest activity yield CLEA was about 16% and 8% for PepX-CLEAs and PepN-CLEAs, respectively. The temperature stability, for example, of the PepX-CLEAs (66% residual activity at 50 °C after 4 d compared to 50% for the free enzyme) and the optimum temperature of PepN-CLEAs (40 °C compared to 30 °C for the free enzyme) increased. Additionally, combi-CLEAs of PepX/PepN were prepared under optimal conditions evaluated for the single CLEAs. Here, the highest activity yield CLEA was about 18% and 9% for PepX and PepN activity, respectively. These combi-CLEAs were applied for hydrolysis into an Alcalase® pre-hydrolyzed casein solution. As proof of principle, it was shown that the combi-CLEAs are suitable for application in protein hydrolysis. The relative degree of hydrolysis was increased by approximately 52% compared to the Alcalase® pre-hydrolyzed casein solution.