Létisse Fabien, Lamare Sylvain, Legoy Marie-Dominique, Graber Marianne
Laboratoire de Génie Protéique et Cellulaire, Pôle Sciences et Technologies, Université de la Rochelle, Bâtiment Marie Curie, Avenue Michel Crépeau, 17042 La Rochelle Cedex 1, France.
Biochim Biophys Acta. 2003 Nov 3;1652(1):27-34. doi: 10.1016/s1570-9639(03)00262-0.
The influence of the addition of an extra component in a gaseous reaction medium, on the kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by immobilized lipase B from Candida antarctica was studied in a continuous solid/gas reactor. In this reactor, the solid phase is composed of a packed enzymatic sample, which is percolated by gaseous nitrogen, simultaneously carrying gaseous substrates and additional components to the enzyme while removing reaction products. The system permits to set thermodynamic activity of all gaseous components (substrates or not) independently at the desired values. This allows in particular to study the influence of an extra added component at a constant thermodynamic activity value, contrary to classical solid/liquid system, which involves large variations of thermodynamic activity of added solvent, when performing full kinetic studies. Alcohol inhibition constant (K(I)) and methyl propionate and propanol dissociation constants (K(MP) and K(P)) have been determined in the solid/gas reactor in the presence of 2-methyl-2-butanol, and compared with values previously obtained in the absence of added component and in the presence of water. Complementary experiments were carried out in the presence of an apolar compound (hexane) and led to the conclusion that the effect of added organic component on lipase-catalyzed alcoholysis is related to their competitive inhibitory character towards first substrate methyl propionate. The comparison of data obtained in liquid or with gaseous 2-methyl-2-butanol shows that lower K(MP) and K(I) are found in gaseous medium, which would correspond on the one hand to a lower acylation rate k(2), and on the other hand to a higher binding rate k(1) between substrate and free enzyme in gaseous medium.
在连续的固/气反应器中，研究了在气态反应介质中添加额外组分对南极假丝酵母固定化脂肪酶B催化丙酸甲酯与正丙醇醇解动力学的影响。在该反应器中，固相由填充的酶样品组成，气态氮对其进行渗透，气态氮在携带气态底物和额外组分至酶的同时，去除反应产物。该系统能够将所有气态组分（无论是否为底物）的热力学活性独立设定为所需值。这尤其允许在恒定的热力学活性值下研究额外添加组分的影响，这与经典的固/液系统相反，在进行完整的动力学研究时，经典固/液系统中添加溶剂的热力学活性会有很大变化。在2-甲基-2-丁醇存在的情况下，在固/气反应器中测定了乙醇抑制常数（K(I)）以及丙酸甲酯和丙醇的解离常数（K(MP)和K(P)），并与之前在无添加组分和有水存在的情况下获得的值进行了比较。在存在非极性化合物（己烷）的情况下进行了补充实验，得出的结论是，添加的有机组分对脂肪酶催化醇解的影响与其对第一种底物丙酸甲酯的竞争性抑制特性有关。对在液体中或与气态2-甲基-2-丁醇获得的数据进行比较表明，在气态介质中发现较低的K(MP)和K(I)，这一方面对应较低的酰化速率k(2)，另一方面对应气态介质中底物与游离酶之间较高的结合速率k(1)。
