1969 Volume 65 Issue 5 Pages 741-750
1. The hydrolysis of phenyl α-maltoside (φM) catalyzed by crystalline saccharifying α-amylase [EC 3. 2. 1. 1] of Bacillus subtilis was studied at various substrate concentrations at 25°C and pH 5.4 in 0.02M acetate buffer by thin layer chromatography, phenol reagent method and modified Somogyi-Nelson's method. The rates of formation of phenol (φ), vφ, phenyl α-glucoside (φG), vφG, and maltotriose (MT), VMT, were deter-mined, and their dependencies on the substrate concentration were investigated. The effect of pH on the rates of formation of φ and φG was examined at a fixed substrate concentration.
2. In the parallel reactions forming φ and φG from φM, vφ follows the Michaelis-Menten equation over the whole range of substrate concentration, s (0.12-120mM). On the other hand, the plot of vφG versus substrate concentration shows a sigmoidal curve at lower substrate concentrations (0.12-10mM), and the plot of s2/vφG versus s gives a straight line, although the rate obeys the Michaelis-Menten kinetics at higher substrate concentrations (11-120mM). The dependency of vφG/vφ on s shows a saturation curve of the Michaelis-Menten type.
3. The optimum pH for the processes forming phenol and phenyl α-glucoside is between 5.3 and 5.4, and the pH values, which give one half the velocity at the opti-mum pH, are 4.0 and 6.75 for formation of phenol, and 3.7 and 7.05 for formation of phenyl α-glucoside.
4. The dependencies of vφ and vφG on the substrate concentration can be explained neither by a simple mechanism of a single active center producing both phenol and phenyl α-glucoside nor by a mechanism of two active centers each forming a single set of products, phenol and maltose or phenyl α-glucoside and glucose. Besides an active site (active center), an activator site to which the substrate is bound is considered necessary for the formation of φG, while such an additional site is not necessary for the formation of phenol.
5. The apparent rate of formation of maltotriose, VMT, was found to be propor-tional to the product of vφG and vφ.
