Extension and application of the “enzyme test bench” for oxygen consuming enzyme reactions

Abstract

Within industrial process development, powerful screening techniques are required to select the optimal biocatalyst regarding such process characteristics as cost effectiveness, turnover number or space time yield. Conventional measurement of the initial enzyme activity, which is the established high throughput screening technique, disregards the long-term stability of an enzyme. A new model based technique called "enzyme test bench" was recently presented before by our group which addresses this issue. It combines the high throughput screening approach with an extensive enzyme characterization, focusing especially on the long-term stability. The technique is based on modeling enzyme activation and deactivation as temperature dependent reactions in accordance with the Arrhenius law. Controlling these reactions by tailor made temperature profiles, the slow long-term deactivation effects are accelerated and characterizing models are parameterized. Thus, the process properties of an enzyme can be predicted and included into the screening procedure. Moreover, the optimum process temperature as function of the envisaged operation time can be found by these means. In this work, the technique is extended to the important class of oxygen consuming reactions. For this aim, a suitable assay and a defined oxygen supply were established. This extended technique was applied to characterize and to optimize a complex, multi-stage laccase-mediator system (LMS). For the variation and optimization of the enzyme to mediator to substrate ratio, experiments in microtiter plates were performed. Predictions from this high throughput characterization were compared to long-term experiments in a RAMOS device (Respiration Activity Monitoring System), a technique for on-line monitoring of the oxygen transfer rate in shake flasks. Within the limits of the model validity, the enzyme test bench predictions are in good agreement with the long-term experiments.

Enzyme activity deviates due to spatial and temporal temperature profiles in commercial microtiter plate readers

“…Several suggestions have been made to overcome edge effects, mostly in a specific constructive way changing the incubation chambers or plates . Commercial MTP readers usually apply external heating units with air as heat transfer medium and feature typically ±0.2 to 0.5°C accuracy according to manufacturer's specifications.…”

Insights into laccase producing organisms, fermentation states, purification strategies, and biotechnological applications

“…For example, Hommes et al modified an extracellular flux analyzer (CFA, originally used to assess physiological parameters in cell monolayers) to estimate the oxygen consumption rates of five types of laccases compared with that of the conventional Clark electrode‐based method. The similar assay protocol was applied for estimation of laccase activity in both free and on fumed silica nanoparticles immobilized form in the investigation conducted by Ammann et al In another study performed by Rachinskiy et al, a novel model‐based technique called an enzyme test bench was applied to characterize and optimize a complex and multi‐stage LMS. They used sodium sulfite, catalyzed by cobalt sulfate, to determine the oxygen transfer rate and the respiration activity monitoring system (RAMOS) technique for the determination of laccase activity …”

“…They used sodium sulfite, catalyzed by cobalt sulfate, to determine the oxygen transfer rate and the respiration activity monitoring system (RAMOS) technique for the determination of laccase activity. 87…”

Influence of the experimental setup on the determination of enzyme kinetic parameters

“…[12][13][14]32 The reproduction of kinetic data is even more challenging for MTPs or heating chambers that have been developed to overcome issues as irregular heating or meniscus formation. 4,[33][34][35][36] Our investigations show that reliable kinetic data are highly dependent on the experimental setup used and highlight the importance to properly state the experimental.…”