Fast determination of biocatalyst process stability

Boy, Matthias; Dominik, Andreas; Voss, Harald

doi:10.1016/s0032-9592(98)00124-1

Cited by 26 publications

(15 citation statements)

References 6 publications

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“…In this case the model was reduced passing on the reversible deactivation mechanism (the respective parameters k R,1 and k R,2 were fixed to the same values). Enzymes directly exhibiting irreversible deactivation from the native state are known from literature (Boy et al, 1999), thus, the model reduction is mechanistically acceptable. The estimated parameters for both esterases together with their standard deviations are presented in Table III.…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, in accordance to Boy et al (1999), the adjustment of the thermodynamic equilibrium between the reversibly deactivated enzyme E R and the active enzyme E N is assumed to be of infinite speed. Thus, it is modeled as an algebraic equation defining the temperature dependent equilibrium constant of reversible enzyme deactivation K R : The deactivation model is completed by the conservation equation:…”

Section: Modelingmentioning

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%

“…With microtiter plates several experiments can be easily conducted in a sequential way. Therefore, the existing approach (Boy et al, 1999) was extended from intuitive experimental design (mostly temperature ramps) to computer aided optimum experimental design regarding the enzyme deactivation model. Experimental design, experiments and parameter estimation are performed in an iterative sequential way, so that experiments, most sensitive to estimated parameters, are conducted to enhance the information content of the measured data.…”

Section: Introductionmentioning

confidence: 99%

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

Rachinskiy

Schultze²,

Boy³

et al. 2009

Biotech & Bioengineering

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Modelingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Rachinskiy

Schultze²,

Boy³

et al. 2009

Biotech & Bioengineering

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show abstract

“…Quantitative assessment of deactivation of enzyme during catalyzed processes is an important, however, often difficult and expensive task [78]. Figure 14 illustrates a direct ultrasonic assessment of stability of β-galactosidase in Cow & Gate First Infant milk at 20 • C during hydrolysis of lactose catalyzed by this enzyme over 24 h. The small concentration of enzyme utilized in the test was chosen to provide small (however measurable) change of concentration of the substrate (3% or 13 cm/s increase in ultrasonic velocity over 24 h), so that the hydrolysis occurred at nearly constant concentration of the substrate (as shown in the inset of Figure 14) [8].…”

Section: Assessment Of Enzyme Deactivation During Long-time Reactionsmentioning

confidence: 99%

Ultrasonic Monitoring of Biocatalysis in Solutions and Complex Dispersions

Buckin

Altas

2017

Catalysts

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Abstract:The rapidly growing field of chemical catalysis is dependent on analytical methods for non-destructive real-time monitoring of chemical reactions in complex systems such as emulsions, suspensions and gels, where most analytical techniques are limited in their applicability, especially if the media is opaque, or if the reactants/products do not possess optical activity. High-resolution ultrasonic spectroscopy is one of the novel technologies based on measurements of parameters of ultrasonic waves propagating through analyzed samples, which can be utilized for real-time non-invasive monitoring of chemical reactions. It does not require optical transparency, optical markers and is applicable for monitoring of reactions in continuous media and in micro/nano bioreactors (e.g., nanodroplets of microemulsions). The technology enables measurements of concentrations of substrates and products over the whole course of reaction, analysis of time profiles of the degree of polymerization and molar mass of polymers and oligomers, evolutions of reaction rates, evaluation of kinetic mechanisms, measurements of kinetic and equilibrium constants and reaction Gibbs energy. It also provides tools for assessments of various aspects of performance of catalysts/enzymes including inhibition effects, reversible and irreversible thermal deactivation. In addition, ultrasonic scattering effects in dispersions allow real-time monitoring of structural changes in the medium accompanying chemical reactions.

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Glucosylglycerol and glucosylglycerate as enzyme stabilizers

Sawangwan

Goedl

Nidetzky

2010

Biotechnology Journal

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Compatible solutes constitute a diverse class of low-molecular-mass organic molecules that are accumulated in high intracellular concentrations in response to the external stress of hyperosmolality or high temperature. Many of these compounds like alpha, alpha-trehalose are well known for their stabilizing effect on protein structure and could lead to development of more stable protein formulations. Negatively charged solutes like mannosylglycerate (R-2-O-alpha-D-mannopyranosyl-glycerate) are widespread among (hyper)thermophilic microorganisms and are thought to be exceptionally potent stabilizers of proteins under high-temperature denaturation conditions. To further inquire into the role of compound charge for protective function, we have compared two naturally occurring and structurally related solutes, glucosylglycerol (2-O-alpha-D-glucopyranosyl-sn-glycerol) and glucosylglycerate (R-2-O-alpha-D-glucopyranosyl-glycerate), as stabilizers of different enzymes undergoing inactivation through elevated temperature or freeze drying, and benchmarked their effects against that of alpha,alpha-trehalose. Glucosylglycerate in concentrations of >/=0.1 M was the most effective in preventing thermally induced loss of enzyme activity of lactate dehydrogenase, mannitol dehydrogenase, starch phosphorylase, and xylose reductase. alpha,alpha-Trehalose could usually be replaced by glucosylglycerol without compromising enzyme stability. Glucosylglycerol and glucosylglycerate afforded substantial (eightfold) protection to mannitol dehydrogenase during freeze drying.

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Fast determination of biocatalyst process stability

Cited by 26 publications

References 6 publications

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

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

Ultrasonic Monitoring of Biocatalysis in Solutions and Complex Dispersions

Glucosylglycerol and glucosylglycerate as enzyme stabilizers

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