酵素反應速度に對する温度効果の解析並びに絶對最適温度に就て : 其3 : 細菌α-Amylaseの場合

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Studies on the effect of temperature upon the rate of dextrinization catalyzed by the α-amylase of Bs. amylosolvens TERUI, and on some other properties of the enzyme were undertaken.Partly purified enzyme preparation having the activity of 4×10^5〜15×10^5 units of WOHLGEMUTE (D^<40._30'>) was used in our experiment : The results are summarized as follows.a) The time, t', necessary to undergo hydrolysis of a definite fraction of the glycosidic bonds in the substrate (soluble starch), can be determined by teh method of iodine coloration. The definite iodine-color corresponds to the definite amount of reducing substances formed per unit of initial substrate concentration, X_0 (Fig. 26,p.372). In the stadium of dextrinization (the period before "just colorless" with iodine), t' is found to be a linear function of X_0 (fig. 1,p.372) in accordance with the expression derived from the MICHAELIS-MENTEN hypothesis, that is t'=1/(kE){X-0(1-r)-Km/nr], …(3)in which r(=X/X_0) is the residual fraction of the substrate and is fixed in a set of experiments by the method of iodine coloration.b) The course of hydrolysis in the stage of post-dextrinization includes some unknown factors. In addition to the already known facts, we point out the persistent difference in the hydrolysis levels with experiments in which temperature is taken as the only variable (fig.3,p.373). The dissociation constant of enzyme-[intermediate hydrolyzate]-complex seems to take conspicuously high value in that stage (cf. the hydrolysis level at 1/2 Vmax in fig.2a and 3).c) The optimum pH for dextrinization is 5.5 (with phosphate buffer, cf. fig.4,p.375), which is somewhat lower than that for the liquefaction of starch.d) The destruction of the enzyme in the course of dextrinization was examined with the aid of 1/t' : E_0-diagram and it was compared with the theoretical diagram drawn on the assumption that the monomolecular destruction constant (Z) of the enzyme is hold unchanged in one set of experiments, where E_0 is taken as the only variable. If the value of Z is independent to E_0,as in ordinary cases, the following relationship, with which it would become possible to compute the value of Z, should be hold, that is Z=1/(t-1)/n(E-<II>)/(E_I-E_II)…(2) where E_1 is the initial concentration of the enzyme, with which t' is t_1,and E_II is that with which t' is 2t_1.An analysis of the discrepance between practical and theoretical diagram (cf. fig.5 and 4), leads us to the conclusion that there appears, in the course of dextrinization, an intermediate hydrolysate exeriting powerful protective action against the enzyme destruction, and that the smaller the value of E_0 is, the stronger is the action. Such a type of protective action seems to be peculier to the dextrinization period. The enzyme is most stable in the pH-range of 5〜6.5.e) The effect of temperature upon the rate constant of dextrinization ware investigated in the range from 0° up to 80℃ under the selected conditions, on which enzyme destruction is quite missible.At higher temeprature, the enzyme was protected by the addition of 0.1% CaCl_2,which was proved to have practically missible effect upon the heat of activation of dextrinization. The temperature coefficienct of the second term in the right hand of eq. (1) was neglected in our computation, for the temperature coefficient of dextrinization at X_0=0.4〜0.45 and 0.8〜0.9was practically equal, and so we adopted the simplified relationship, [numerical formula]…(3)The activation energy (μ) and the heat of activation (ΔH^*) were calculated as shown in table 1 (p. 380). The (ΔH^*)exp. stands in linear relationship with T and applying the method of least square we obtain (Δ^*)e×p・=-0.25T+1.24_s(kilo col.)…(4)Therefrom, the absolute optimum (T_AO), where (ΔH^*exp) becomes-RT, is found to be 61._3℃ (334._4°K).f) If the third hypothesis on the theory of T_AO would be correct, the equillibrium constant, Kai between reactive from and r
公益社団法人日本生物工学会の論文
1950-09-15

酵素反應速度に對する温度効果の解析並びに絶對最適温度に就て : 其3 : 細菌α-Amylaseの場合

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