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

Kunze M, Lattermann C, Diederichs S, Kroutil W, Büchs J - J Biol Eng (2014)

Bottom Line: Microtiter 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: AVT-Chair for Biochemical Engineering, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany.

ABSTRACT

Background: Bioprocesses 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.

Results: In 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.

Conclusion: Microtiter 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.

No MeSH data available.


Related in: MedlinePlus

Enzymatic hydrolysis of 4-methylumbelliferyl-ß-D-cellobioside (4MUC) with cellulase from T. reseei (Celluclast) applying a temperature profile in a MTP. (A) Progresses of 4-methyl-umbelliferyl (4MU) fluorescence (λex = 365 nm, λem = 455 nm) during the hydrolysis of 4MUC at different temperatures. Online data of 12 (from 48) exemplary wells. (B) Calibration curve for fluorescence intensity at varied 4MU concentration. (C) Temperature dependent initial reaction rate and (D) resulting Arrhenius plot of Celluclast. Dotted lines indicate Arrhenius fit. Experimental conditions: 96well MTP, VL = 200 μL, n = 995 rpm, d0 = 3 mm, 1 g/L Celluclast and 0.6 mM 4MUC in 0.1 M acetate buffer, pH = 4.8. Temperature profile: RT = 37°C, Tset,low = 10°C, Tset,high = 95°C (comp. Figure 3E).
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Figure 9: Enzymatic hydrolysis of 4-methylumbelliferyl-ß-D-cellobioside (4MUC) with cellulase from T. reseei (Celluclast) applying a temperature profile in a MTP. (A) Progresses of 4-methyl-umbelliferyl (4MU) fluorescence (λex = 365 nm, λem = 455 nm) during the hydrolysis of 4MUC at different temperatures. Online data of 12 (from 48) exemplary wells. (B) Calibration curve for fluorescence intensity at varied 4MU concentration. (C) Temperature dependent initial reaction rate and (D) resulting Arrhenius plot of Celluclast. Dotted lines indicate Arrhenius fit. Experimental conditions: 96well MTP, VL = 200 μL, n = 995 rpm, d0 = 3 mm, 1 g/L Celluclast and 0.6 mM 4MUC in 0.1 M acetate buffer, pH = 4.8. Temperature profile: RT = 37°C, Tset,low = 10°C, Tset,high = 95°C (comp. Figure 3E).

Mentions: As a candidate for the optimization of enzymatic reactions the commercial cellulase cocktail Celluclast was chosen since it is often used for biomass degradation. The Celluclast cocktail contains a mixture of several cellulases from the fungus Trichoderma reesei[48]. In order to follow the enzyme reaction on-line, a substrate, namely 4-methylumbelliferyl-β-D-cellobioside (4MUC), was used. It releases the fluorescent dye 4-methylumbelliferone (4MU) when hydrolyzed by cellulases which can be easily detected with a fluorescence spectrometer. Consequently, the 4MUC assay is commonly used for the high-throughput screening of cellulolytic enzymes [49]. Compared to the microbial systems described before, a higher temperature optimum was expected for the cellulases. Consequently, a profile was chosen providing a higher temperature range of 42-65°C (comp. Figure 3E).Twelve exemplary curves resulting from the 4MUC hydrolysis by Cellucalst are depicted in Figure 9A, whereby, the fluorescence intensity indicates the formation of the product 4MU. For all conditions the typical course of enzymatic reactions is observed with a strong increase of the product concentration in the beginning which runs into saturation after a certain time when no substrate is available anymore. The reaction is strongly temperature dependent. The highest reaction rates are observed at 53.2-56.2°C. Consequently, these curves run into saturation already after approx. 3 h. Furthermore, the highest final product concentrations occur at these temperatures. In the temperature ranges above and below reduced reaction rates and extended reaction times for complete substrate conversion are observed. Pure substrate showed a constant signal close to 0 a.u., thereby, proving that no substrate reacted in the absence of enzyme. In order to quantify the cellulolytic reaction, the 4MU fluorescence signal was calibrated assuming that each cleaving of a 4MUC molecule releases one 4MU molecule (Figure 9B). In this way reaction rates as well as final product concentrations can be calculated.


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

Kunze M, Lattermann C, Diederichs S, Kroutil W, Büchs J - J Biol Eng (2014)

Enzymatic hydrolysis of 4-methylumbelliferyl-ß-D-cellobioside (4MUC) with cellulase from T. reseei (Celluclast) applying a temperature profile in a MTP. (A) Progresses of 4-methyl-umbelliferyl (4MU) fluorescence (λex = 365 nm, λem = 455 nm) during the hydrolysis of 4MUC at different temperatures. Online data of 12 (from 48) exemplary wells. (B) Calibration curve for fluorescence intensity at varied 4MU concentration. (C) Temperature dependent initial reaction rate and (D) resulting Arrhenius plot of Celluclast. Dotted lines indicate Arrhenius fit. Experimental conditions: 96well MTP, VL = 200 μL, n = 995 rpm, d0 = 3 mm, 1 g/L Celluclast and 0.6 mM 4MUC in 0.1 M acetate buffer, pH = 4.8. Temperature profile: RT = 37°C, Tset,low = 10°C, Tset,high = 95°C (comp. Figure 3E).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4128537&req=5

Figure 9: Enzymatic hydrolysis of 4-methylumbelliferyl-ß-D-cellobioside (4MUC) with cellulase from T. reseei (Celluclast) applying a temperature profile in a MTP. (A) Progresses of 4-methyl-umbelliferyl (4MU) fluorescence (λex = 365 nm, λem = 455 nm) during the hydrolysis of 4MUC at different temperatures. Online data of 12 (from 48) exemplary wells. (B) Calibration curve for fluorescence intensity at varied 4MU concentration. (C) Temperature dependent initial reaction rate and (D) resulting Arrhenius plot of Celluclast. Dotted lines indicate Arrhenius fit. Experimental conditions: 96well MTP, VL = 200 μL, n = 995 rpm, d0 = 3 mm, 1 g/L Celluclast and 0.6 mM 4MUC in 0.1 M acetate buffer, pH = 4.8. Temperature profile: RT = 37°C, Tset,low = 10°C, Tset,high = 95°C (comp. Figure 3E).
Mentions: As a candidate for the optimization of enzymatic reactions the commercial cellulase cocktail Celluclast was chosen since it is often used for biomass degradation. The Celluclast cocktail contains a mixture of several cellulases from the fungus Trichoderma reesei[48]. In order to follow the enzyme reaction on-line, a substrate, namely 4-methylumbelliferyl-β-D-cellobioside (4MUC), was used. It releases the fluorescent dye 4-methylumbelliferone (4MU) when hydrolyzed by cellulases which can be easily detected with a fluorescence spectrometer. Consequently, the 4MUC assay is commonly used for the high-throughput screening of cellulolytic enzymes [49]. Compared to the microbial systems described before, a higher temperature optimum was expected for the cellulases. Consequently, a profile was chosen providing a higher temperature range of 42-65°C (comp. Figure 3E).Twelve exemplary curves resulting from the 4MUC hydrolysis by Cellucalst are depicted in Figure 9A, whereby, the fluorescence intensity indicates the formation of the product 4MU. For all conditions the typical course of enzymatic reactions is observed with a strong increase of the product concentration in the beginning which runs into saturation after a certain time when no substrate is available anymore. The reaction is strongly temperature dependent. The highest reaction rates are observed at 53.2-56.2°C. Consequently, these curves run into saturation already after approx. 3 h. Furthermore, the highest final product concentrations occur at these temperatures. In the temperature ranges above and below reduced reaction rates and extended reaction times for complete substrate conversion are observed. Pure substrate showed a constant signal close to 0 a.u., thereby, proving that no substrate reacted in the absence of enzyme. In order to quantify the cellulolytic reaction, the 4MU fluorescence signal was calibrated assuming that each cleaving of a 4MUC molecule releases one 4MU molecule (Figure 9B). In this way reaction rates as well as final product concentrations can be calculated.

Bottom Line: Microtiter 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: AVT-Chair for Biochemical Engineering, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany.

ABSTRACT

Background: Bioprocesses 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.

Results: In 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.

Conclusion: Microtiter 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.

No MeSH data available.


Related in: MedlinePlus