Studies of immobilized and cross-linked a[alpha]-chymotrypsin to explore solvent stabilization

Abstract

Immobilized enzyme post-column reactors in HPLC systems yield good sensitivity and selectivity in biochemical analyses. The problem, however, is that the mobile phase, usually methanol based, readily denatures the enzyme column unless a dilution system is employed prior to this component. The dilution element not only makes the entire system cumbersome, but also introduces various conditions that complicate the procedure. This paper describes an immobilization study that used intramolecular and intermolecular cross-linking techniques to examine whether enzymes retain their catalytic activity when exposed to high methanol concentrations as in HPLC systems. An investigation of the activity of several immobilized enzyme columns with respect to methanol concentration were performed. The activities of free, nonimmobilized, immobilized, and intra- and intermolecularly cross-linked a.-chymotrypsin were studied in increasing concentrations of methanol by monitoring the hydrolysis rate ofN-benzoyl-L-tyrosine ethyl ester (BTEE) at 256nm. Plots of activity vs. methanol concentration were done to determine the stability of the free, immobilized, and immobilized/cross-linked enzymes in methanol. The resultant activity plots revealed that the free enzyme yielded very little activity in a 50% methanol environment, while the immobilized enzyme column demonstrated maximum activity in 60%-70% methanol matrices. Intramolecular cross-linking techniques that were applied to the immobilized enzyme lead to inactive columns. The intermolecular techniques proved to be more successful since activity was observed in these columns up to and including 100% methanol.