Product Distribution in Amylase-Catalyzed Hydrolysis of Amylose<subtitle>Comparison of Experimental Results with Theoretical Predictions</subtitle> (1980)

1. Hydrolyses of amylose with a number-average degree of polymerization (⁠DP¯n) of 17 by single amylase system and mixed amylase system containing endo- and exo-type amylases were quantitatively followed by the measurement of total reducing power, determination of glucose using the glucose oxidase-peroxidase method and of oligosaccharides by high performance liquid chromatography, and the fluorescence method using a fluorescent dye, 2-p-toluidinylnaphthalene-6-sulfonate. The amylases used in this study were Taka-amylase A (TAA), bacterial liquefying α-amylase (BLA), and glucoamylase (GA) from Rhizopus niveus. All the reactions were carried out at pH 5.1 and 25°C. 2. It has been indicated that change in product distribution with time can be predicted theoretically for the amylase-catalyzed reaction by using a small number of parameters (subsite parameters), based on the subsite theory (Matsuno, R. et al. (1978) J. Biochem. 83, 385–394; Kondo, H. et al. (1978) J. Biochem. 84, 403–417). It was found that this theoretical treatment could be successfully applied to the mixed amylase system by taking into account the “simple hydrolysis,” disregarding transglycosylation, condensation, and multiple attack. 3. The following results were obtained by comparing experimental results with theoretical predictions. a) Single amylase system: 1) Experimental product distribution curves of oligosaccharides with degrees of polymerization (DP's) of 1∼7 were quite similar to theoretical ones which took into account simple hydrolysis. The product distribution curves differed significantly from amylase to amylase depending on their subsite structures. 2) The experimental extent of reaction as a function of time (time course) was quite similar to the theoretical prediction in the single amylase system of TAA and GA. 3) The theoretical product distribution curves which took into account simple hydrolysis were slightly but significantly different from experimental results in the cases of TAA and GA. Namely, glucose was produced in a larger amount in the single TAA system and short chain products (DP=2∼7) were produced in much more significant amounts at the initial stage of reaction in the single GA system than was predicted by the theory. These can be reasonably accounted for, at least in part, by considering multiple attack in addition to simple hydrolysis; in the former the enzyme can attack the same substrate molecule successively several times before allowing the substrate molecule to dissociate. 4) The contribution of multiple attack in the TAA and GA reactions was also indicated in the fluorescence value-extent of reaction plot (Kondo, H. et al. (1978) J. Biochem. 84, 403–417). b) Mixed amylase system: This system includes the following two cases: the case in which TAA and GA attacked simultaneously the substrate molecule (“simultaneous system”) and the case in which only TAA was used for the hydrolysis of substrate up to the extent of reaction of 10.5% and thereafter only GA was used (“sequential system”). For both cases, satisfactory agreement was obtained between the experimental product distribution curves and time course and theoretical ones based on simple hydrolysis alone. It was found experimentally that the time required to reach the extent of reaction of 90% was prolonged by about 3 times in the simultaneous system of TAA and GA and by 1.6 times in the sequential system of TAA and GA as compared to the single GA system. These were successfully reproduced by theoretical predictions.