A New Method for Rapid Determination of Biocatalyst Process Stability

Boy, Matthias; Böhm, Dirk; Voss, Harald

doi:10.3109/10242420108992027

Cited by 8 publications

(7 citation statements)

References 5 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A comprehensive assessment of biocatalyst stability under process conditions at constant temperature is experimentally rather demanding as-in principle-this would require operation of the biocatalyst under process conditions (high substrate concentrations) for the required operation time at least for a small set of different temperatures (Boy et al, 2001). A more efficient approach is the estimation of the temperature-dependent deactivation of the enzyme by a model-based experimental analysis (Bommarius and Broering, 2005;Boy et al, 2001).…”

Section: Estimation Of Biocatalysts Process Stabilitymentioning

confidence: 99%

“…A more efficient approach is the estimation of the temperature-dependent deactivation of the enzyme by a model-based experimental analysis (Bommarius and Broering, 2005;Boy et al, 2001). This requires foremost an accurate reactor and enzyme kinetics model that needs to be expanded in order to capture the temperature-dependent activation of the biochemical reaction (Eq.…”

Section: Estimation Of Biocatalysts Process Stabilitymentioning

confidence: 99%

“…In order to estimate the parameters for enzyme activation and deactivation, we used a model-based experimental analysis approach based on a least square method (Matlab, The MathWorks, Natick, MA) applying the experimentally determined degree of conversion in an EMR operated at defined temperatures and fed with a solution containing one of the two methionine enantiomers at constant composition (Boy et al, 2001). The applied model consisted of the reversible Michaelis-Menten kinetics of the CFE (Eqs.…”

Section: Enzyme Activation and Deactivation Parameter Estimationmentioning

confidence: 99%

See 2 more Smart Citations

Model‐based characterization of an amino acid racemase from Pseudomonas putida DSM 3263 for application in medium‐constrained continuous processes

Bechtold

Makart

Reiss

et al. 2007

Biotech & Bioengineering

View full text Add to dashboard Cite

The amino acid racemase with broad substrate specificity from Pseudomonas putida DSM 3263 was overproduced and characterized with respect to application in an integrated multi-step process (e.g., dynamic kinetic resolution) that--theoretically--would allow for 100% chemical yield and 100% enantiomeric excess. Overexpression of the racemase gene in Escherichia coli delivered cell free extract with easily sufficient activity (20-50 U mg(-1) total protein) for application in an enzyme membrane reactor (EMR) setting. Model-based experimental analysis of a set of enzyme assays clearly indicated that racemization of the model substrates D- or L-methionine could be accurately described by reversible Michaelis-Menten kinetics. The corresponding kinetic parameters were determined from progress curves for the entire suitable set of aqueous-organic mixtures (up to 60% methanol and 40% acetonitrile) that are eligible for an integrated process scheme. The resulting kinetic expression could be successfully applied to describe enzyme membrane reactor performance under a large variety of settings. Model-based calculations suggested that a methanol content of 10% and an acetonitrile content of 20% provide maximum productivity in EMR operations. However product concentrations were decreased in comparison to purely aqueous operation due to decreasing solubility of methionine with increasing organic solvent content. Finally, biocatalyst stability was investigated in different solvent compositions following a model-based approach. Buffer without organic content provided excellent stability at moderate temperatures (20-35 degrees C) while addition of 20% acetonitrile or methanol drastically reduced the half-life of the racemase.

show abstract

Section: Estimation Of Biocatalysts Process Stabilitymentioning

confidence: 99%

Section: Estimation Of Biocatalysts Process Stabilitymentioning

confidence: 99%

Section: Enzyme Activation and Deactivation Parameter Estimationmentioning

confidence: 99%

See 1 more Smart Citation

Model‐based characterization of an amino acid racemase from Pseudomonas putida DSM 3263 for application in medium‐constrained continuous processes

Bechtold

Makart

Reiss

et al. 2007

Biotech & Bioengineering

View full text Add to dashboard Cite

show abstract

“…Figure 1b shows the applied model scheme of enzyme deactivation. Considering different deactivation mechanisms from the literature (Baptista et al, 2003;Boy et al, 2001;Collins et al, 2003;Daniel et al, 1996;Hei and Clark, 1993;Jurado et al, 2004;Nikolova et al, 1997;Peterson et al, 2004;Polakovic and Bryjak, 2002;Polakovic and Vrabel, 1996), four enzyme fractions are defined: active enzyme, reversibly deactivated enzyme, irreversibly deactivated enzyme, irreversibly deactivated agglomerates. The arrows in the figure depict the change from fraction to fraction as chemical reactions with reaction constants k, which are assumed to behave in accordance to the Arrhenius law.…”

Section: Temperature Controlmentioning

confidence: 99%

“…These long-term experiments are not compatible with high throughput screening approaches. Boy et al (1999Boy et al ( , 2001 and Gibbs et al (2005) propose an elegant approach to determine the long term enzyme stability within a short time. Similar to the breaking tests employed in material science, the enzyme behavior is observed under partly extreme conditions to predict the long term behavior under more moderate operation conditions.…”

Section: Introductionmentioning

confidence: 99%

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

Rachinskiy

Schultze²,

Boy³

et al. 2009

Biotech & Bioengineering

View full text Add to dashboard Cite

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.

show abstract

Entwicklung eines Enzymstabilitätstests mit hohem Durchsatz

Rachinskiy

Büchs

Boy

et al. 2006

Chemie Ingenieur Technik

View full text Add to dashboard Cite

bedingungen parallel zu testen. Durch die Modulform der Membranfestbettreaktoren ist in der weiteren Prozessentwicklung ein einfaches Scale-up möglich.Aufgrund der gewählten Immobilisierungsbedingungen und der verwendeten Membranen werden die Enzyme reversibel gebunden. Bei einem Verlust der Enzymaktivität oder bei einem gewünschten Wechsel der Enzymaktivität, kann das bereits immobilisierte Enzym schnell und vollständig von der Membran eluiert werden. Nach der Reaktivierung der Membran kann die erneute Enzymbindung erfolgen. Die eingesetzten Membranen zeigen sich für nachfolgende Immobilisierungen gleiche Bindungskapazitäten. Das vorgestellte Verfahren ermöglicht eine zeitsparende Prozessentwicklung.

show abstract

A New Method for Rapid Determination of Biocatalyst Process Stability

Cited by 8 publications

References 5 publications

Model‐based characterization of an amino acid racemase from Pseudomonas putida DSM 3263 for application in medium‐constrained continuous processes

Model‐based characterization of an amino acid racemase from Pseudomonas putida DSM 3263 for application in medium‐constrained continuous processes

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

Entwicklung eines Enzymstabilitätstests mit hohem Durchsatz

Contact Info

Product

Resources

About