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

Abstract

BackgroundBioprocesses depend on a number of different operating parameters and temperature is one of the most important ones. Unfortunately, systems for rapid determination of temperature dependent reaction kinetics are rare. Obviously, there is a need for a high-throughput screening procedure of temperature dependent process behavior. Even though, well equipped micro-bioreactors are a promising approach sufficient temperature control is quite challenging and rather complex.ResultsIn this work a unique system is presented combining an optical on-line monitoring device with a customized temperature control unit for 96 well microtiter plates. By exposing microtiter plates to specific temperature profiles, high-throughput temperature optimization for microbial and enzymatic systems in a micro-scale of 200 μL is realized. For single well resolved temperature measurement fluorescence thermometry was used, combining the fluorescent dyes Rhodamin B and Rhodamin 110. The real time monitoring of the microbial and enzymatic reactions provides extensive data output. To evaluate this novel system the temperature optima for Escherichia coli and Kluyveromyces lactis regarding growth and recombinant protein production were determined. Furthermore, the commercial cellulase mixture Celluclast as a representative for enzymes was investigated applying a fluorescent activity assay.ConclusionMicrotiter plate-based high-throughput temperature profiling is a convenient tool for characterizing temperature dependent reaction processes. It allows the evaluation of numerous conditions, e.g. microorganisms, enzymes, media, and others, in a short time. The simple temperature control combined with a commercial on-line monitoring device makes it a user friendly system.

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

“…S6). For both devices, Synergy Mx and Synergy 4, the average temperatures show increasing standard deviation of ±0.8 to 1.3°C and ±0.3 to 0.9°C, respectively with increasing temperatures, which corresponds well to typical standard deviations of ±0.5 to 0.9°C at 37°C over the whole plate [13,20,30,32].…”

“…The temperature‐dependent enzyme activities were fitted by an Arrhenius equation extended by a Hougen–Watson expression for the temperature optimum of the catalyst (Eq. ) . …”

“…The temperature-dependent enzyme activities were fitted by an Arrhenius equation extended by a Hougen-Watson expression for the temperature optimum of the catalyst (Eq. 3) [32,50]. Therein, the numerator contains the constant (A) and the activation energy (E a ), the denominator contains the con-stant (B) and the free energy of the denaturation reaction (ΔG) (Supporting information, Table S3).…”

Modelling the enzyme catalysed substrate conversion in a microbioreactor acting in continuous flow mode

“…The substrate concentrations (31) and (32) in the dimensionless form are expressed in a slightly more closed form,…”

Estimating the success of enzyme bioprospecting through metagenomics: current status and future trends

“…The identification depends heavily on gene and protein abundance, and although we are aware that a large part of the transcriptome and proteome remains unseen, it can be assumed that the identified genes and enzymes might represent the predominant (in terms of dosage per cell and expression levels) and the most active genes and enzymes under the tested conditions. A further evaluation of enzyme performance under multiple conditions using high‐throughput parameter (Kunze et al ., ) may allow sorting out the possibility to identify highly active, efficient and promiscuous (Pandya et al ., ) enzymes under real or close‐to‐real process conditions, independently of the further optimization phase to which the enzyme can be subjected (Bornscheuer et al ., ).…”