TY - JOUR

T1 - Proteolytic ceramic capillary membranes for the production of peptides under flow

AU - Antink, Marieke M.Hoog

AU - Sewczyk, Tim

AU - Kroll, Stephen

AU - Árki, Pál

AU - Beutel, Sascha

AU - Rezwan, Kurosch

AU - Maas, Michael

N1 - Funding Information: The authors are grateful for funding by the DFG through grant KR 3802/5-1 and to Novozymes A/S, Denmark, for providing the Alcalase® 2.5.

PY - 2019/7/15

Y1 - 2019/7/15

N2 - In this study, we investigate the effect of membrane surface functionalization on the immobilization of the protease subtilisin A and its performance in the production of peptides from the model protein casein under flow. The surface of tubular ceramic membranes was silanized to yield carboxylated and aminated supports for enzyme immobilization via non-covalent and carbodiimide activated binding. The protease density correlated with electrostatic interactions between the positively charged enzyme and the supports, with the highest enzyme density reached on negatively charged, carboxylated membranes (0.019 molecules/nm², noncovalent approach). Enzyme leaching was reduced by covalent binding of protease to carboxylated supports (5% leached) and slightly improved by binding to aminated membranes (46%) over non-covalent binding to unfunctionalized reference capillaries (66%). Regarding carbodiimide activated immobilization, protease on unfunctionalized and aminated supports exhibited a significantly larger specific activity (0.99 μmol/min/mg) than enzymes on carboxylated surfaces (0.15 μmol/min/mg), which suggests preferred enzyme orientation. In protein hydrolysis, these differences in surface-enzyme interactions were reflected by variations in peptide composition and degree of hydrolysis. Accordingly, we demonstrate that surface functionalization critically determines the surface properties of protease support materials for the production of peptides under flow and allows tailoring the performance of proteolytic capillary membranes.

AB - In this study, we investigate the effect of membrane surface functionalization on the immobilization of the protease subtilisin A and its performance in the production of peptides from the model protein casein under flow. The surface of tubular ceramic membranes was silanized to yield carboxylated and aminated supports for enzyme immobilization via non-covalent and carbodiimide activated binding. The protease density correlated with electrostatic interactions between the positively charged enzyme and the supports, with the highest enzyme density reached on negatively charged, carboxylated membranes (0.019 molecules/nm², noncovalent approach). Enzyme leaching was reduced by covalent binding of protease to carboxylated supports (5% leached) and slightly improved by binding to aminated membranes (46%) over non-covalent binding to unfunctionalized reference capillaries (66%). Regarding carbodiimide activated immobilization, protease on unfunctionalized and aminated supports exhibited a significantly larger specific activity (0.99 μmol/min/mg) than enzymes on carboxylated surfaces (0.15 μmol/min/mg), which suggests preferred enzyme orientation. In protein hydrolysis, these differences in surface-enzyme interactions were reflected by variations in peptide composition and degree of hydrolysis. Accordingly, we demonstrate that surface functionalization critically determines the surface properties of protease support materials for the production of peptides under flow and allows tailoring the performance of proteolytic capillary membranes.

KW - Bioactive peptides

KW - Casein digestion

KW - Enzyme immobilization

KW - Membrane bioreactor

KW - Subtilisin A

KW - Surface functionalization

UR - http://www.scopus.com/inward/record.url?scp=85064228826&partnerID=8YFLogxK

U2 - 10.1016/j.bej.2019.04.005

DO - 10.1016/j.bej.2019.04.005

M3 - Article

VL - 147

SP - 89

EP - 99

JO - Biochemical engineering journal

JF - Biochemical engineering journal

SN - 1369-703X

ER -