Linking the Pre-steady-state, Steady-state, and Zero-order kinetic parameters together for Industrial and allied applications

Abstract

Background: There is an increasing application of enzymes to achieving the industrial goal of the production of biomolecules of different kinds relevant to food, textiles, pharmaceuticals, automobiles, environmental remediation, etc.; there appears 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. This ought to be a prerequisite for industrial (batch or reactor) design. Objectives: The study is carried out with the objectives of deriving equations that can be explored in relating pre-zero-order (otherwise known as pre-steady-state (prss)) to zero-order kinetic parameters, such as the Michaelis-Menten constant (KM), maximum velocity of catalysis (Vmax), and specificity constant (SC), and ultimately evaluating the equations with experimental data. Methods: The study was theoretical and experimental, backed by the Bernfeld method of generating initial rates. Results and discussion: The Vmax for the higher industrial-type concentration of the enzyme (alpha-amylase) is 7812.5 micromoles/L/min, while the KM is 115.1 g/L. The SC obtained by calculation, either by the new equation (Eq. (25b)) or the ratio Vmax:KM, is 67.88 micromoles L/g min, and surprisingly, as against the literature, the SC obtained by a new graphical method is 275.4 micromoles L/g min using sub-KM values of substrate concentrations. The prss Vmax and KM are 2348.62 ± 479.94 micromoles L/g min and 7.41 ± 1.77 g/L. Conclusion: The need to derive an equation linking prss and the zero-order kinetic parameter is justified by the result obtained. In order to establish consistency, 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.

Turnover and Catalytic cycle frequency Determination based on Molar mass-dependent Model equations

“…The preparation of reagents and methods (Bernfeld) of assay for the generation of initial rates and the determination of kinetic parameters were as previously described [23].…”

“…( 28) assumes the same structure as Eq. ( 29), but different methods and equations are needed for the determination of k1 [22,23]. Equation (28)…”

“…As in the recent literature [23], Aspergillus oryzae alpha-amylase (EC 3.2.1.1) and insoluble potato starch were purchased from Sigma-Aldrich, USA. Tris 3, 5-dinitrosalicylic acid, maltose, and sodium potassium tartrate tetrahydrate were purchased from Kem Light Laboratories in Mumbai, India.…”