Kinetics and Mechanism of Transfer Action of Saccharifying α-Amylase of Bacillus subtilis

Abstract

1. The formation of phenol (φ) was observed in the mixture of maltose (M) and phenyl α-glucoside (φG), which was catalyzed by saccharifying α-amylase (EC 3. 2. 1. 1) of Bacillus subtilis, although φG itself was not hydrolyzed either in the presence or absence of glucose. Thin layer chromatography (TLC) of the reaction mixture revealed the formation of a small amount of phenyl α-maltoside (φM). From these results, it was concluded that φM was produced from M and φG by glucosyl transfer catalyzed by the enzyme, and then φM was hydrolyzed into φ and M. Since the overall rate of formation of phenol, vφ, is limited by the rate of transfer, vr, the latter can be obtained directory from the measurement of the former. This system was utilized for studying the kinetics of transfer action of this enzyme. 2. The rate of transfer as a function of φG concentration at a fixed concentration of M proceeded according to the Michaelis-Menten kinetics within the range of concentrations of φG examined, and the apparent Michaelis constant KmφG for φG was 250 mM at a maltose concentration of 42 mM. On the other hand, the rate of transfer, vr, as a function of the concentration of M in a fixed concentration of φG showed a remarkable sigmoidal shape in the region of lower substrate concentrations and showed a maximum at around 100 mM, above which it decreased hyperbolically. 3. The ratio between the rate of transfer, vT, and that of hydrolysis of maltose, vW, vT/vW increased with the concentration of φG, showing that the hydrolysis of M is inhibited by φG. 4. Methyl alcohol was found to serve as an acceptor, judging from the formation of methyl α-glucoside detected by TLC. 5. The hydrolysis of M, and the transfer action of a glucosyl residue of M to φG and to M were explained by assuming essentially the same mechanism as that previously proposed for the hydrolysis of φM.