Bousquet-Dubouch M P, Graber M, Sousa N, Lamare S, Legoy M D
Université de la Rochelle, Laboratoire de Génie Protéique et Cellulaire, Pôle Sciences et Technologies, Avenue Crépeau, 17042 La Rochelle, Cedex 1, France.
Biochim Biophys Acta. 2001 Nov 26;1550(1):90-9. doi: 10.1016/s0167-4838(01)00273-4.
The kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by Candida antarctica lipase B supported onto silanized Chromosorb P was studied in a continuous solid/gas reactor. In this system the solid phase is composed of a packed enzymatic sample and is percolated by nitrogen as carrier gas, which simultaneously carries substrates to the enzyme while removing reaction products. In this reactor the thermodynamic activity of substrates and effectors can be perfectly adjusted allowing kinetic studies to be performed under different operating conditions. The kinetics obtained for alcoholysis were suggested to fit a Ping Pong Bi Bi mechanism with dead-end inhibition by the alcohol. The values of all apparent kinetic parameters were calculated and the apparent dissociation constant of enzyme for gaseous ester was found very low compared with the one obtained for liquid ester in organic medium, certainly due to the more efficient diffusion in the gaseous phase. The effect of water thermodynamic activity was also investigated. Water was found to act as a competitive inhibitor, with a higher inhibition constant than n-propanol. Thus alcoholysis of gaseous methyl propionate and n-propanol catalyzed by C. antarctica lipase B was found to obey the same kinetic mechanism as in other non-conventional media such as organic liquid media and supercritical carbon dioxide, but with much higher affinity for the substrates.
在连续固/气反应器中研究了负载在硅烷化Chromosorb P上的南极假丝酵母脂肪酶B催化丙酸甲酯与正丙醇的醇解动力学。在该系统中，固相由填充的酶样品组成，并由氮气作为载气渗透，氮气在携带底物至酶的同时去除反应产物。在该反应器中，可以完美调节底物和效应物的热力学活性，从而能够在不同操作条件下进行动力学研究。醇解反应的动力学被认为符合乒乓双底物双产物机制，存在醇的终产物抑制作用。计算了所有表观动力学参数的值，发现酶对气态酯的表观解离常数与在有机介质中液体酯的表观解离常数相比非常低，这肯定是由于在气相中扩散更有效。还研究了水的热力学活性的影响。发现水作为竞争性抑制剂，其抑制常数高于正丙醇。因此，发现南极假丝酵母脂肪酶B催化的气态丙酸甲酯与正丙醇的醇解遵循与其他非传统介质（如有机液体介质和超临界二氧化碳）相同的动力学机制，但对底物具有更高的亲和力。
