A General Guide for the Optimization of Enzyme Assay Conditions Using the Design of Experiments Approach

Abstract

Many factors must be considered during the optimization of an enzyme assay. These include the choice of buffer and its composition, the type of enzyme and its concentration, as well as the type of substrate and concentrations, the reaction conditions, and the appropriate assay technology. The process of an enzyme assay optimization, in our experience, can take more than 12 weeks using the traditional one-factor-at-a-time approach. In contrast, the design of experiments (DoE) approaches have the potential to speed up the assay optimization process and provide a more detailed evaluation of tested variables. However, not all researchers are aware of DoE approaches or believe that it is easy to employ a DoE approach for the optimization of an assay. In order to facilitate enzyme assay developers to use DoE methodologies, we present in detail the steps required to identify in less than 3 days (1) the factors that significantly affect the activity of an enzyme and (2) the optimal assay conditions using a fractional factorial approach and response surface methodology. This is exemplified with the optimization of assay conditions for the human rhinovirus-3C protease, and the methodology used could be employed as a basic guide for the speedy identification of the optimum assay conditions for any enzyme.

Optimization of a Colorimetric Assay to Determine Lactate Dehydrogenase B Activity Using Design of Experiments

“…When we run the assay using these assay conditions, the kinetic parameters K M , V max , and k cat were found to be equal to (i) for NAD+: 376.3 ± 22.3 µM, 881.2 ± 44.2 nmol/min/mL, and 32.6×10 –3 s –1 , respectively; and (ii) for lactate: 23.3 ± 1.23 mM, 891.0 ± 37.5 nmol/min/mL, and 32.9 ×10 –3 s –1 ; a Z′ factor of 0.75 and an S/N of 16.8 were obtained. In our laboratory, we have recently developed a DoE-based screening and optimization approach 27 to identify the best assay conditions of the human rhinovirus 3C protease (HRV-3CP). Therefore, in this study, we subsequently carried out a similar approach to examine whether alteration of assay conditions could improve the activity of LDH-B, as described in the following sections.…”

“…1) (see Ref. 27 and references cited therein). In a half-normal plot, all significant factors are away from zero, while the nonsignificant (unimportant) factors have a normal distribution centered close to zero.…”

“…The statistical parameter Z-prime (Z′) was used to assess the quality of the optimized assay. 27 The Z′-factor was calculated based on Equation (4):…”

“…In contrast, statistical design of experiment (DoE) approaches provide information on variable interactions that cannot be obtained by the trial-and-error approaches, and they have been successfully used for the optimization of several assays (see Ref. 27 and references cited therein). The theory and potential applications of DoE approaches are extensively described in many excellent textbooks 28,29 (for a review on the topic, see Ref.…”

“…To this end, several DoE software packages, including Design Expert (Stat-Ease Inc., Minneapolis, MN), JMP (SAS Institute Inc., Cary, NC), Minitab (Minitab, University Park, PA), and ECHIP (ECHIP, Wilmington, DE), are available to facilitate the choice of the DoE, the design of the experiments, and to interpret the results. Even though a lot of experimenters believe that statistically designed experiments are difficult to use, 27,30 these DoE software packages require only basic knowledge of statistics and can help a DoE novice during the design of experimental assay conditions and interpretation of the results. 30…”