糸状菌の生産するセルラーゼに関する研究 : (第2報) ペニシリウムセルラーゼの動力学的解析

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As the cellulase system contained sufficient amounts of enzymes by which soluble intermediates formed were degraded quickly to glucose, the wieight loss of wood pulp powder (cf. preceding report) corresponded to the increase of glucose in the reaction mixture. Such a system was convenient for the kinetic study with native cellulose as subsrate, especially in the case where a soluble product was inhibitory. Time course of reaction could be followed by estimating either glucose or residual insoluble substrate.The effect of substrate concentration upon reaction velocity was investigated either by changing initial substrate concentration [S]_0 or by following residual substrate concentration [S]. Unexpectedly, the results were shown to obey the Michaelis-Mentent rule. When [S] was followed, fitness to the Michaelis equation was tested with the expression obtained by integrating the original equation with respect to time, and the time required for reducing [S] at any time to γ[S] was plotted against [S], where the factor γ(<1) was fixed at any value (e.g. 0.8). Pre-incubation of the substrate with the enzyme solutio at a temperature low enough to stop the reaction had almost no effect in enhancing the initial reaction rate.These results could be interpreted by assuming that neither the adsorption rate nor penetratio of enzyme moleucle was limiting the overall rate of substrate dissolution, and that the overall density of points in the substrate open for the chance of enzymic attack was maintained at a level almost steady throughout the reaction time. Thus the effective substrate concentration could be expressed by α[S] instedad of [S], where α could be regarded as constant. Modified Michaselis-Menten equation was obtained by substituting [S] by α[S], which had essentially the same form as the original one. The above points having almost equal chance of being attacked by the enzyme, were regarded to distribute in the outer part of amorphous region, and if so, the distiribution would be steep owing to the existence of penetration barrier. Modified Michaelis constant K_m' (=K_m/α) had a value of ca. 3.57g/l, which was obtained from initial velocity experiments.The reaction was shown to be inhibited by the final product. Analysis of the time course of reaction indicated that the inhibition was of competitive nature, and that the inhibitor concentration [I] was increased as the reaction proceeded owing to the accumulation of glucose, which was proportional to [S]_0-[S], i.e.[I]=c([S]_0-[S]). In analyzing the time course was used an integrated form of amended Michaelis equation in which the effect of increasing inhibitor concentration was involved. The value of K_i was deduced from the above analysis to be 1.54c.
社団法人日本生物工学会の論文
1965-05-25

糸状菌の生産するセルラーゼに関する研究 : (第2報) ペニシリウムセルラーゼの動力学的解析

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