“Enzyme Test Bench,” a high‐throughput enzyme characterization technique including the long‐term stability

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 homogenous temperature control in the range of 10-85 +/- 0.5 degrees C. A universal fluorescent assay for online acquisition of ester hydrolysis reactions by pH-shift is developed and established. The developed instrumentation and assay are applied to characterize two esterases. The results of the characterization, carried out in microtiter plates applying short term experiments of hours, are in good agreement with the results of long term experiments at different temperatures in 1 L 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.

Minireactor-based high-throughput temperature profiling for the optimization of microbial and enzymatic processes

“…The here used measurement setup is a modification of an earlier developed system, the so called Enzyme Test Bench [4,9]. It combines an optical on-line monitoring system for cultivations and reactions in microtiter plates with a special system for temperature regulation.…”

“…Thus, the wells are jacketed by these radiators providing efficient heat transfer between the thermostating block and the MTP wells. The heat transfer properties of the system were specified before [4]. The whole system itself was placed in a temperature controlled environment.…”

“…The design of the heating block is based on the work of Rachinskiy et al [4,9]. It is made of aluminium with tube adapters for the two water circulation systems (Figure 1C).…”

“…reaction rates or yields [1]. Thereby, the temperature optimum can be different within one microbial or enzymatic system, either the focus is on growth or product formation [2], on enzyme activity or stability [3,4]. …”

“…microtiter plates (MTP), in temperature controlled rooms [7] or incubators [8]. Another option is to link the MBR system to a thermostat system and circulate water through the MBR chamber base [4,9-11]. The major disadvantage of these systems is their limitation in operating parallel reactors at different temperatures.…”