Rapid determination of enzyme purity by a microdialysis-based assay

Abstract

Microdialysis was shown to be useful as a fast on-line sampling method for determining the purity of starch hydrolysing enzymes. The enzymes were characterised using their hydrolytic properties. β-Amylases and pullulanases from different sources and/or manufacturers were investigated, with maltose, maltoheptaose, pullulan, and potato amylopectin starch (PAP) as substrates. The hydrolysis products were sampled via an on-line microdialysis probe and determined in a high-performance anion-exchange chromatographic (HPAEC) system. Comparison between the expected (theoretical) hydrolysis products with those obtained in the experiments made it possible to determine impurities in the enzymes. Two of the β-amylases and one pullulanase released unwanted hydrolysis products, indicating trace impurities in the enzyme preparation. Microdialysis sampling allows on-line sampling and eliminates separate sample preparation and clean-up steps. On-line microdialysis coupled to a HPAEC system is therefore a fast and simple technique for analysing enzyme hydrolysates.