Automated flow-injection procedures for the determination of hydrolytic enzymes in bioreactor preparations

Summary

Automated spectrophotometric flow-injection (FI) procedures for the quantitative determination of the hydrolytic enzymes amylase, xylanase, polygalacturonase and protease, acting on macromolecular substrates, are described. The peptides produced by the protease are derivatized with trinitrobenzene sulphonic acid. For the other enzymes, the reducing sugars produced are derivatized with p-aminobenzoylhydrazide (PABH). The FI manifold design allows the choice of any required detetion, range between 0.1 U ml⁻¹ and several hundred U ml⁻¹. Two FI manifold designs are proposed; one optimizes, sample throughput at a high sensitivity level by incorporating several parallel incubation coils, the other minimizes sample volume at a low sensitivity level and facilitates, automation. The instrumentation is largely based on commercial HPLC equipment.