Carbonyl reductase of Candida parapsilosis – Stability analysis and stabilization strategy

For industrial applications, there is a demand for larger diversity of similar well-characterized enzymes in order to test them for a given process (biodiversity screening). For fundamental science, the comparison of enzymes with similar function but different sequence can provide insight into structure function relationships or the evolution of enzymes. This study gives a good example on how this demand can be efficiently met.

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“…; Grosch et al . ). Assessment of more stable, predominantly thermostable, enzymes has therefore been attempted with different strategies.…”

Section: Introductionmentioning

confidence: 97%

Discovery of a novel thermostable Zn²⁺ -dependent alcohol dehydrogenase from Chloroflexus aurantiacus through conserved domains mining

et al. 2018

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“…Also transketolase, another ThDP‐dependent enzyme, inactivates during reaction progress in the presence of the substrate . Fortunately, the biocatalyst stability can be improved by enzyme engineering and reaction engineering . As an example, continuous membrane reactors maintain substrate concentration at low level and thereby, circumvent substrate dependent inactivation …”

Section: Introductionmentioning

confidence: 99%

Simultaneous identification of reaction and inactivation kinetics of an enzyme‐catalyzed carboligation

Ohs

Leipnitz

Schöpping

et al. 2018

Biotechnology Progress

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Thiamine diphosphate (ThDP)-dependent enzymes catalyze a broad range of reactions with excellent enantioselectivity. Among these reactions, carboligations of aldehydes are of particular interest since the products, chiral hydroxy ketones, are valuable building blocks in the pharmaceutical industry. However, the substrates, for example, benzaldehyde, inactivate the biocatalysts, for example the ThDP-dependent benzaldehyde lyase from Pseudomonas fluorescens (PfBAL). Because only few mechanistic kinetic models for carboligation and simultaneous inactivation are available today, we quantitatively determined the reaction kinetics and inactivation of the self-carboligation of benzaldehyde yielding the product (R)-benzoin catalyzed by PfBAL directly from progress curves using model-based experimental analysis. Discrimination of several inactivation models identified the substrate-dependent inactivation by benzaldehyde to be significant. Sensitivity analysis and optimal experimental design improved parameter precision significantly, to between 4 and 26% relative standard deviation while maintaining the necessary number of 13 experiments moderate. The developed mechanistic kinetic model will enable to perform a model-based process optimization to circumvent the substrate-dependent enzyme inactivation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1081-1092, 2018.

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“…Enzyme production was performed in terrific broth (TB) medium (pH 7.0, 12 g L −1 tryptone, 24 g L −1 yeast extract, 5 g L −1 glycerol, 12.54 g L −1 K 2 HPO 4 , 2.31 g L −1 KH 2 PO 4 , 0.1 g L −1 ampicillin and 0.1 g L −1 MgCl 2 ). Pre‐cultures were inoculated by cryo cultures (25% v/v glycerol) and carried out in 10 mL TB medium in 250 mL shake flasks (350 rpm, 50 mm shaking diameter, 37°C) using RAMOS technology . Pre‐cultures were harvested at an oxygen transfer rate (OTR) of about 50 mmol L −1 h −1 .…”

Section: Methodsmentioning

confidence: 99%

“…Protein concentration was determined by Bradford and bicinchoninic acid protein assay. To reduce the amount of interfering substances, the enzyme solution was diluted with deionized water and purified by ultrafiltration unit (Sartorius, Vivaspin 6 10000 MWCO PES) . The final enzyme concentration was adjusted to 5.5 mg mL −1 by addition of triethanol amine (TEA, 50 mmol L −1 , pH 7.0, 1 mmol L −1 MgCl 2 ) buffer and stored at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…Precultures were inoculated by cryo cultures (25% v/v glycerol) and carried out in 10 mL TB medium in 250 mL shake flasks (350 rpm, 50 mm shaking diameter, 378C) using RAMOS technology. 25 Pre-cultures were harvested at an oxygen transfer rate (OTR) of about 50 mmol L 21 h 21 . Fermentation was carried out in a BIOSTATV R B plus fermenter (Sartorius Stedim Biotech, G€ ottingen, Germany) at 378C with a starting OD 600 of 0.1.…”

Section: Enzyme Production and Purificationmentioning

confidence: 99%

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Influence of the experimental setup on the determination of enzyme kinetic parameters

Grosch

Wagner

Knaup

et al. 2016

Biotechnology Progress

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For the design of bioconversion processes parallel experimentation in microtiter plates is commonly applied to reduce the experimental load, although data accuracy and reproducibility are often reduced. In an effort to quantify the impact of different microscale experimental systems on the estimation of enzyme kinetic parameters from progress curves, we comprehensively evaluated the enzymatic reduction of acetophenone in both open and closed polystyrene and quartz microtiter plates as well as quartz cuvettes. Differences in conversion of up to 50% over time were observed increasing from polystyrene MTPs to quartz MTPs to quartz cuvettes. Initial reaction velocities increased systematically from polystyrene to quartz MTPs and cuvettes. The experimental errors decreased in the same order showing highest experimental error of about 20% in polystyrene. We further evaluated reasons causing the deviations within one system as well as between the systems. The choice of reaction vessel material, temperature effects and substrate cross contaminations in MTPs were shown to be of importance in the experimental results. Although the experimental data differed between the reaction vessels, no distinct trends in estimated kinetic parameters were found. While the microkinetic parameters vary up to an order of magnitude between different systems, the corresponding macrokinetic parameters lie in the same range for all systems varying by 29-118%. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:87-95, 2017.

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Carbonyl reductase of Candida parapsilosis – Stability analysis and stabilization strategy

Cited by 11 publications

References 41 publications

Discovery of a novel thermostable Zn²⁺ -dependent alcohol dehydrogenase from Chloroflexus aurantiacus through conserved domains mining

Discovery of a novel thermostable Zn²⁺ -dependent alcohol dehydrogenase from Chloroflexus aurantiacus through conserved domains mining

Simultaneous identification of reaction and inactivation kinetics of an enzyme‐catalyzed carboligation

Influence of the experimental setup on the determination of enzyme kinetic parameters

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Carbonyl reductase of Candida parapsilosis – Stability analysis and stabilization strategy

Cited by 11 publications

References 41 publications

Discovery of a novel thermostable Zn2+ -dependent alcohol dehydrogenase from Chloroflexus aurantiacus through conserved domains mining

Discovery of a novel thermostable Zn2+ -dependent alcohol dehydrogenase from Chloroflexus aurantiacus through conserved domains mining

Simultaneous identification of reaction and inactivation kinetics of an enzyme‐catalyzed carboligation

Influence of the experimental setup on the determination of enzyme kinetic parameters

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Product

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Discovery of a novel thermostable Zn²⁺ -dependent alcohol dehydrogenase from Chloroflexus aurantiacus through conserved domains mining

Discovery of a novel thermostable Zn²⁺ -dependent alcohol dehydrogenase from Chloroflexus aurantiacus through conserved domains mining