Linking the Pre-Steady-State, Steady-State, and Zero-Order Kinetic Parameters Together for Industrial Applications

Abstract

There appeared not to have been a way of linking pre-zero-order kinetics to zero-order kinetics so as to garner key kinetic parameters at very high industrial concentrations of the substrate. The objectives were the derivation of equations that can be explored in relating pre-zero-order (pre-steady-state (prss)) to zero-order kinetic parameters (ZOK), such as the Michaelis-Menten constant (KM), maximum velocity of catalysis (Vmax), and specificity constant (SC), to be evaluated. The Vmax for the higher industrial-type concentration of the enzyme (alpha-amylase) was 7812.5 micromoles/l/min, while the KM was 115.1 g/l. The SC obtained by calculation, either by the new equation (Eq. (25b)) or the ratio Vmax:KM, was 67.88 micromoles l/g min. Surprisingly, as compared to the literature, the SC obtained by the new graphical method was 275.4 micromoles l/g min using sub-KM values of substrate concentrations. The prss Vmax and KM were 2348.62 ± 479.94 micromoles l/g min and 7.41 ± 1.77 g/l, respectively. There is a justification for an equation linking PRSS and the ZOK, which can enhance reactor design. The equation linking PRSS to the ZOK kinetic parameters was derived. With the equation, the KM for a very high industrial concentration of the substrate and the enzyme that would have been impossible was made possible. Future studies may focus on assays at high concentrations of the enzyme and sub-KM concentrations of the substrate so as to observe a repeat of higher SC. Note that if the concentration of one enzyme is twice (or more) the concentration of another enzyme of the same kind, the Vmax of the first should be twice the Vmax of the latter given saturating concentrations of the substrate for each enzyme; the parameter that is constant is the catalytic first-order rate constant (kcat); it may be theoretically assumed that the KM can follow the same order as with the Vmax. Apart from a very high concentration of the gelatinized starch that retards the mobility of the enzyme, the spectrophotometer has an upper limit of its power to read color development.