A coupled enzyme nylon tube reactor pyruvate kinase-lactate dehydrogenase system

Abstract

A coupled enzyme nylon tube reactor has been made by simultaneously immobilizing rabbit muscle pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) onto the inside surface of a nylon tube initially derivatized with poly (scl)-lysine to serve as a spacer molecule. The enzymes were covalently linked to the amino groups of the poly lysine spacer molecule by cross-linking with glutaraldehyde. The coupled enzyme system may be used in routine analysis to determine the concentrations of PEP, ADP, pyruvate, and NADH in a given solution and also to modify radioactively labeled nucleotides. The kinetic properties of the enzymes appear to be partially diffusion controlled as shown by an inverse relationship ofK m(app) values and activities to the flow rate of substrates through the tube reactor. This coupled enzyme system may be used as an indicator system when used in conjunction with other enzymes to complete a sequence of catalytic steps. An example of this is demonstrated in this paper by linking a nylon tube supported acetate kinase (EC 2.7.2.1) to this coupled enzyme system so that the three enzymes function in a series that facilitates the estimation of acetate.