Suitability and limitations of methods for characterisation of activity ofmalto-oligosaccharide-forming amylases

The suitability and limitations of essential methods and reference substrat es used for characterisation of activity of amylolytic enzymes is investiga ted. Saccharogenic, chromogenic and chromatographic methods are included. T he results are discussed in relation to the measurement of reaction rates, determination of action mode and product specificity and the impact on iden tification and nomenclature of malto-oligosaccharide-forming amylases. An a ccurate determination of reaction rates using the saccharogenic methods str ongly depends on the degree of polymerisation (DP) of the standards used an d the hydrolysis products formed by the amylase. Particularly the use of gl ucose as standard can lead to overestimates due to the differences in the r educing potential of glucose and malto-oligosaccharides. The reliability of the chromogenic methods for determination of action mode depends on the DP of the substrate and the specificity of the amylase. For a characterisatio n of the starch hydrolysis products and the variation in the DP during hydr olysis, high performance anion-exchange chromatography with pulsed amperome tric detection provided a fast and reliable method. A literature survey rev ealed varying and inconsistent use of nomenclature of malto-oligosaccharide forming amylases. Therefore a systematic approach identifying three main c lasses of activity is suggested using not only the mode of action and the D P of the major product but also the stage of hydrolysis at which this produ ct is formed. (C) 2000 Elsevier Science Ltd. All rights reserved.