['To understand the intrinsic role of hydrolytic enzymes in sludge treatment, particularly their effect on the digestibility and dewaterability of sludge, activated sludge flocs were disintegrated using various techniques that included different enzymes (amylase, cellulase, proteinase, DNase, and polygalacturonase), pH adjustment, and temperature adjustment. The effectiveness of each enzyme treatment was pinpointed by quantifying the spatial distribution of each type of organic matters (protein, polysaccharide, DNA, fluorescent organics) in outer layer extracellular polymeric substances (EPS), inner layer EPS, and cells.', 'Most hydrolytic enzymes functioned only owing to a temperature or pH effect. The release of organic matter from the interior fraction of EPS to the exterior fraction was prompted under high pH and temperature even without enzyme addition. The effectiveness of enzyme addition was only significant for cellulase and polygalacturonase treatments. Polygalacturonase unexpectedly increased the total EPS polysaccharides up to seven times, accompanied with improved dewaterability, while the amount of EPS proteins was almost unchanged. Combining chemical and morphological evidence, a new conceptual model considering the spatial distribution of polygalacturonic acid-like matter, proteins, cellulose, and other organics in EPS was proposed.'] Does enzyme disintegration with spatial resolution reveal different distributions of sludge extracellular polymer substances?