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2 : 麥芽α-Amylaseの場合

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Regular behavior to the substrate concentration and temperature were observed with malt α-amlyase, partly purified after destroying β-amylase. As to the substrate concentration, there exists the relationship, t'=aX_0+b, …(1) where t' is the time required for the hydrolysis of the substrate to a definite degree, which is indicated by iodine color, X_0 is the initial substrate concentration, and α and β are the constants.We have adopted properly the iodine color corresponding to the reducing value of 16.4 mg of maltose per 100 mg of soluble starch. Eq. (1) can be derived from the well-known MICHAELIS-MENTEN hypothesis, namely -dX/dt=kE_0X/(Km+X), …(2) in which -dX/a't is the rate of the reaction at substrate concentration X. By integrating the above equation from t=o to t=t', we obtain l'=1/(kE_0){X_0(1-r)-Kmlnr}, …(3) where r equals X/X_0 at the time t', and therefore r would have a fixed value. It is easily seen from eq. (1) and (3), that β/α=-Kmlnr/(1-r). If enzyme destruction takes place, eq. (3) must be amended as k∅'=X_0(1-r)-Kmlur, …(4) whre &empty' is the integrated activity of the enzyme throughout the reaction time (t_z) required to attain to that iodine color when Z>O. Hence, it follows that t'/t_s=(p-1)/lnp, …(5) in which p=E/E_0,provided that the enzyme destruction is monomolecular. The above relationships are shown in fig. 5 and 6 (p.295). <OM>^^^- in fig. 6 corresponds to Kmtnr/(1-r). The time-temperature range where Z is practically zero, can be ascertained graphically and the evaluation of Z is also made possible. Table 1 shows the results with reaction mixtures containing phosphate buffer (pH5.4) and small amount of Ca↚. Linear relationships were maintained up to 70℃, and remarkable inactivation took place at 75℃, where Z was evaluated as 0.12 [1/min.]. Computation was made of the activation energy using the equation : μ=RT_1T_2/(ΔT)lnQ ; Q=k_2/(k_1)=Δt_1'/(Δt_2'), …(6) in which Δt'1 is the time value, (t' for X_0+ΔX_0)-(t' for X_0) at T_1 Δt'_2 is that at a higher temterature T. and ΔT⪇. ΔH^* in table 2 is found to be a linear function of T, and formulated by the method of least square, that is ΔH^*=0.57_7T+194._7 (in kilo cal. unit). Therefore, the absolute optimum (T^AO) would be 65._5℃. The lines in fig. 7 were drawn closely to the experimental plots with the assumption that the temperature coefficients of Km equal 1. Insignificance of the variation of Km with temperature is presumable, although accuracy of our experiment was not high enough to estimate the coefficients. Entropy and free energy of activation were calculated on the basis of the above results and the turnover number of the enzyme, reported by SCHWIMMER and BALLS. [table]
社団法人日本生物工学会の論文
1950-07-15

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2 : 麥芽α-Amylaseの場合

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