Sequential ordered, enzymatic reaction

Thus far only reactions involving a single substrate have been considered. Most enzymatic reactions have two substrates. Unlike chemical processes, the sequence in which the substrates bind to the enzyme may be important. If two substrates, A and B, bind in a specific order (e.g., A binds first) as illustrated in Equation 11.37 the mechanism is called ordered sequential. 

Detection launch the detection in Immusoft in order to follow the enzymatic reaction by chrono-amperometry at 250 mV versus Ag/AgCl (sequential detections of 2 s of potential application performed in each channel one after the other). 

Multiple substrate reactions are more frequently occurred than single substrate reactions. Thus, efforts have been made to develop two or more substrate enzymatic reaction mechanisms. One of the most important examples is lipid hydrolysis, where lipid and water molecules act as two substrates to produce two products namely fatty acids and glycerol. Cleland (1963) proposed three types of mechanism for two substrate reactions based on the order of adding the substrates and products release from the active site within the reaction sequence. These are ordered-sequential, random-sequential, and Ping-Pong, shown in Figure 4.1. 

Metabolic pathway 5. Sequential order of enzymatic reactions... 

Since enzymes generally function under the same or similar conditions, several biocatalytic reactions can be carried out in a reaction cascade in a single flask. Thus, sequential reactions are feasible by using multienzyme systems in order to simplify reaction processes, in particular if the isolation of an unstable intermediate can be omitted. Furthermore, an unfavorable equilibrium can be shifted towards the desired product by linking consecutive enzymatic steps. This unique potential of enzymes is increasingly being recognized as documented by the development of multienzyme systems, also denoted as artificial metabolism [15]. 

In order to establish the two-step strategy, both en2ymatic steps were evaluated separately this included the engineering of the TK to accept the non-natural substrate propanal, and use of a bioinformatic-based strategy to identify a suitable co-TA with the ability to accept the ligation product. The final reaction was performed in two sequential steps, because preliminary experiments indicated that the co-TA also aminates the TK substrates in the presence of an amine donor. Notably, for the reductive amination (second step), cheap and achiral 2-PrNH2 could be used successfully as an alternative amine source. The final product was isolated after the two enzymatic steps, with a calculated overall isolated yield of 18% under nonoptimized conditions. However, while the TK-catalyzed C-C bond formation already displayed suitable conversions (23%), the cb-TA was identified to be the bottleneck of the cascade reaction further optimization, especially with respect to the reductive amination, should facilitate a more efficient process. 

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