Entwicklung einer modellbasierten Hochdurchsatztechnik zur Enzymcharakterisierung mit Schwerpunkt auf Langzeitstabilität

• Development of a model based high throughput enzyme characterization technique with a focus on the long term stability

Rachinskiy, Kirill; Büchs, Jochen (Thesis advisor)

Aachen : Publikationsserver der RWTH Aachen University (2008)
Dissertation / PhD Thesis

Aachen, Techn. Hochsch., Diss., 2008

Abstract

A new high throughput technique for enzyme characterization with specific attention to the long term stability, called "Enzyme Test Bench", is presented. The concept of the Enzyme Test Bench consists of short term enzyme tests in 96-well microtiter plates under partly extreme conditions to predict the enzyme long term stability under moderate conditions. The technique is based on the mathematical modeling of temperature dependent enzyme activation and deactivation. Adapting the temperature profiles in sequential experiments by optimal non-linear experimental design, the long term deactivation effects can be purposefully accelerated and detected within hours. During the experiment the enzyme activity is measured online to estimate the model parameters from the obtained data. Thus, the enzyme activity and long term stability can be calculated as a function of temperature. The engineered instrumentation provides for simultaneous automated assaying by fluorescent measurements, mixing and aeration as well as homogenous temperature control in the range of 10-85°C. Fluorescent assays for online acquisition of different reactions, e.g. hydrolysis reactions by pH-shift and oxidation reactions by DOT, are developed and established. The developed instrumentation and assay are applied to characterize two esterases and a complex, multi-stage laccase-mediator system. The results of the enzyme characterization of both esterases, carried out in microtiter plates applying short term experiments of hours, are in good agreement with the results of long term ex-periments at different temperatures in 1L stirred tank reactors of a week. Thus, the new technique allows for both: the enzyme screening with regard to the long term stability and the choice of the optimal process temperature regarding such process parameters as turn over number, space time yield or optimal process duration. The comparison of the temperature dependent behavior of both characterized enzymes clearly demonstrates that the frequently applied estimation of long term stability at moderate temperatures by simple activity measurements after exposing the enzymes to elevated temperatures may lead to suboptimal enzyme selection. Thus, temperature dependent enzyme characterization is essential in primary screening to predict its long term behavior.