Enzyme test bench: a biochemical application of the multirate modeling

Abstract:
In the expanding field of “white biotechnology” enzymes are frequently applied to catalyze the biochemical reaction from a resource material to a valuable product. Designed by evolution to catalyze the metabolism in any life form, they selectively accelerate complex reactions under physiological conditions. Modern techniques, such as directed evolution, have been developed to satisfy the increasing demand on enzymes. Applying these techniques and also rational protein design, the improvement of enzymes’ activity, selectivity and stability is the aim. To tap the full potential of these techniques, it is essential to combine them with adequate screening methods. Nowadays a great number of high throughput colorimetric and fluorescent enzyme assays are applied to measure the initial enzyme activity with high throughput. However, the prediction of enzyme long term stability within short experiments is still a challenge. 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 conducted under partially 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 optimum nonlinear 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 results of the characterization, carried out in micro liter format applying short term experiments of hours, are in good agreement with the long term experiments of a week carried out in 1L stirred tank reactors. 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. This article gives a successful example for the application of multi-rate modeling, experimental design and parameter estimation within biochemical engineering. At the same time, it shows the limitations of the methods at the state of the art and brings the current problems to the attention of the applied mathematics community.

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