A quenched-flow system for measuring heterogeneous enzyme kinetics with sub-second time resolution

Publication date: Available online 13 June 2017 Source:Enzyme and Microbial Technology Author(s): Johan P. Olsen, Jeppe Kari, Kim Borch, Peter Westh Even though many enzyme processes occur at the interface of an insoluble substrate, these reactions are generally much less studied than homogenous enzyme reactions in the aqueous bulk. Interfacial (or heterogeneous) enzyme reactions involve several reaction steps, and the established experimental approach to elucidate multi-step reactions is transient (or pre steady-state) kinetics. A key requirement for pre steady-state measurements is good time resolution, and while this has been amply achieved in different commercial instruments, they are generally not applicable to precipitating suspensions of insoluble substrate. Perhaps for this reason, transient kinetics has rarely been reported for heterogeneous enzyme reactions. Here, we describe a quenched-flow system using peristaltic pumps and stirred substrate suspensions with a dead time below 100ms. The general performance was verified by alkali catalyzed hydrolysis of 2,4-dinitrophenyl acetate (DNPA), and the applicability to heterogeneous reactions was documented by two cellulases (Cel7A and Cel7B) acting on suspensions of microcrystalline cellulose (Avicel) at different loads up to 15g/l. The results showed distinctive differences between the two enzymes. In particular, we found that endo-lytic Cel7B combined very quickly with the substrate and reached the maximal activit...
Source: Enzyme and Microbial Technology - Category: Biotechnology Source Type: research
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