An investigation of the action of porcine pancreatic alpha-amylase on native and gelatinised starches

The action of pancreatic alpha -amylase (EC 3.2.1.1) on various starches ha s been studied in order to achieve better understanding of how starch struc tural properties influence enzyme kinetic parameters. Such studies are impo rtant in seeking explanations for the wide differences reported in postpran dial glycaemic and insulinaemic indices associated with different starchy f oodstuffs. Using starches from a number of different sources, in both nativ e and gelatinised forms, as substrates for porcine alpha -amylase, we showe d by enzyme kinetic studies that adsorption of amylase to starch is of kine tic importance in the reaction mechanism, so that the relationship between reaction velocity and enzyme concentration [E-0] is logarithmic and describ ed by the Freundlich equation. Estimations of catalytic efficiencies were d erived from measurements of k(cat)/K-m performed with constant enzyme conce ntration so that comparisons between different starches were not complicate d by the logarithmic relationship between E-0 and reaction velocity. Such s tudies reveal that native starches from normal and waxy rice are slightly b etter substrates than those from wheat and potato. After gelatinisation at 100 degreesC, k(cat)/K-m values increased by 13-fold (waxy rice) to 239-fol d (potato). Phosphate present in potato starch may aid the swelling process during heating of suspensions; this seems to produce a very favourable sub strate for the enzyme. Investigation of pre-heat treatment effects on wheat starch shows that the relationship between treatment and k(cat)/K-m is not a simple one. The value of k(cat)/K-m rises to reach a maximum at a pre-tr eatment temperature of 75 degreesC and then falls sharply if the treatment is conducted at higher temperatures. It is known that amylose is leached fr om starch granules during heating and dissolves. On cooling, the dissolved starch is likely to retrograde and become resistant to amylolysis. Thus the catalytic efficiency tends to fall. In addition, we find that the catalyti c efficiency on the different starches varies inversely with their solubili ty and we interpret this finding on the assumption that the greater the sol ubility, the greater is the likelihood of retrogradation. We conclude that although cx-amylase is present in high activity in digestive fluid, the enz ymic hydrolysis of starch may be a limiting factor in carbohydrate digestio n because of factors related to the physico-chemical properties of starchy foods. (C) 2001 Elsevier Science B.V. All rights reserved.