Enzymatic synthesis kinetic parameters

In the kinetic approach, the serine or cysteine peptidase rapidly reacts with a suitable acyl donor ester to form the acylenzyme intermediate, which can be deacylated competitively by the added nucleophilic amine component and water. The ratio between aminolysis and hydrolysis of the acyl donor ester is of great importance for the outcome of the synthesis route.This selectivity is essentially determined by the S subsite specificity of the enzyme as shown above. To establish an optimum synthesis strategy, it is useful to know the basic kinetic parameters for the reaction course, in particular those obtained by S subsite mapping are of great importance for planning and optimization of the enzymatic synthesis. 

Gonsalves LR, Femandez-Lafuente R, Guisan IM et al. (2002) The role of 6-aminopenicillanic acid on the kinetics of amoxiciUin enzymatic synthesis catalyzed by peniciUin G acylase immobilized onto glyoxyl agarose. Enzyme Microb Technol 31 464-471 Gorziglia G, Altamirano C, Conejeros R et al. (2002). Determination of kinetic parameters in the synthesis of ampicillin with immobilized penicillin acylase. Annals of the XV Chilean Congress of Chemical Engineering, Punta Arenas, October 2002, pp 107-112. 

Although it is not necessary to determine kinetic parameters in order to use enzymes for chemical synthesis, this knowledge can often be useful in dedding which avenues offer the best opportunities for process improvements. Likewise, it is not essential - but often highly useful - to understand the mechanism of the enzyme-catalyzed reaction for the same reasons that one often benefits from knowing the mechanisms of any reaction employed in a synthetic route. Although every individual enzymatic reaction has a uitique combination of kinetic properties and reaction mechanism, several useful generalizations are summarized in the subsequent section. 

For the in vitro screening of enzyme pairs and enzymatic pathways, immobilized enzyme microreactor systems (lEMR) were established. This includes the reversible immobilization of His -tagged enzymes via nickel-nitriloacetic acid (Ni-NTA) linkage on the surface-derivatized silica. First of all the kinetic parameters for a new enzymatic pathway have to be defined via a transketolase and a w-transaminase in a continuous flow system for a two-step asymmetric synthesis of chiral amino alcohols [147]. The apparenf value is found to be flow rate dependent with different flow rates ranging from 2 to 30 j1/min. The turnover rate was also found to be lower than that in solu-... 

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