Gudrun Gygli

Storage of thermophysical properties (e.g. density, viscosity, thermodynamic activity of water, conductivity) using Chemical Markup Language (CML).

Investigation 1: An experimental workflow to provide detailed information of the molecular mechanisms of enzymes is described. This workflow will help in the application of enzymes in technical processes by providing crucial parameters needed to plan, model and implement biocatalytic processes more efficiently. These parameters are homogeneity of the enzyme sample (HES), kinetic and thermodynamic parameters of enzyme kinetics and binding of reactants to enzymes. The techniques used to measure ...

Because enzyme activity depends very much on the reaction conditions, it is crucial to report all these metadata (see for example the STRENDA Guidelines:https://www.beilstein-strenda-db.org/strenda/public/guidelines.xhtml).

Another challenge in experiments to determine enzyme reaction parameters is the choice of suitable substrate concentrations to enable optimal kinetic fits and the informed choice of a kinetic model.

A Jupyter notebook is given to assist in the choice of substrate concentrations ...

An experimental workflow to provide detailed information of the molecular mechanisms of enzymes is described. This workflow will help in the application of enzymes in technical processes by providing crucial parameters needed to plan, model and implement biocatalytic processes more efficiently. These parameters are homogeneity of the enzyme sample (HES), kinetic and thermodynamic parameters of enzyme kinetics and binding of reactants to enzymes. The techniques used to measure these properties are ...

This is a collection of deep eutectic solvent (DES) experimental and simulation data that is stored in CML format and analysed using gradient boosting decision trees.

This study briefly shows how a Progress Curve (Time-Course) Analysis can look like.

This study briefly shows how a Selwyn test can be performed.

TBD

This study explores how to design an initial rate experiment. It starts with a "zero-round" experiment, which is used to design a "first-round" experiment, which then leads to the design of a "gold-round" experiment.

Measure Gre2p activity by following the change in NADPH absorbance at 340 nm for the conversion of different substrates.

Measure binding of reactants to Gre2p by ITC (isothermal titration calorimetry).

Measure kinetics of reactants by Gre2p with ITC (isothermal titration calorimetry).

Measure homogeneity of an enzyme sample (Gre2p) with DLS (dynamic light scattering).

A meta-analysis of the impact of water content and temperature on the viscosities of four deep eutectic solvents (glyceline, reline, DEAG, DEACG), their components (choline chloride, urea, glycerol, ethylene glycol), methanol, and pure water was performed. We analyzed the viscosity data by an automated workflow, using Arrhenius and Vogel–Fulcher–Tammann-Hesse models.

Determination of thermophysical properties (densities, diffusion coefficients, energy contributions) of different methanol-water mixtures using molecular dynamics simulations.

This is a collection of data involving choline chloride:glycerol:water mixtures stored in CML.

This is a collection of data that have been used to analyse data on deep eutectic solvent mixtures of choline chloride:glycerol:water.

Preparation needed to use Simulation Foundry, Version 1.5.

Please read the manual before working with this Simulation Foundry.

Pay careful attention to the installation instructions.

Note the known issues.

This is the simulation part of the Simulation Foundry, Version 1.5.

Download and unpack the zip file and the .sh bash script. Copy them into a folder which has a meaningful name. Launch the bash script, following the instructions in the manual.

Make sure you followed the instructions in "Preparation" before running this.

This is the analysis part of the Simulation Foundry, Version 1.5.

Download and unpack the zip file and the .sh bash script. Copy them into a folder which has a meaningful name. Launch the bash script, following the instructions in the manual.

Make sure you followed the instructions in "Preparation" before running this.

Here would be the results from the Analysis part of the Simulation foundry. For the newest version, which was improved based on reviewers comments to represent best practices, of the Simulation Foundry for Methanol-Water mixtures no such results exist yet.

Homogeneity of the Gre2p samples in KPi Buffer before and after different treatments, including different amounts of tween-20 in the buffer (0.01, 0.1, 1.0%).

Specific activity of Gre2p measured by following the change in absorbance of NADPH at 340 nm for the conversion of nitrononane-2,8-dione (NDK).

Kinetic parameters (Km, kcat) of Gre2p measured by following the change in absorbance of NADPH at 340 nm for the conversion of nitrononane-2,8-dione (NDK) or hexane-2,5-dione. Initial rates at different substrate concentrations are measured.

Specific activity of Gre2p measured by following the change in absorbance of NADPH at 340 nm for the conversion of nitrononane-2,8-dione (NDK) using different enzyme concentrations.

ITC binding (BIND) experiment to determine the binding parameters of NADPH to Gre2p in 100 mM KPi Buffer.

ITC binding (BIND) experiment to determine the binding parameters of NADPH to Gre2p in 1x PBS Buffer.

ITC binding (BIND) experiments to determine the binding parameters of NADPH to Gre2p in 100 mM HEPES Buffer.

ITC binding (BIND) experiments to determine the binding parameters of NADP+ to Gre2p in 100 mM KPi Buffer.

ITC binding (BIND) experiments to determine the binding parameters of NADP+ to Gre2p in 1x PBS Buffer.

ITC binding (BIND) experiments to determine the binding parameters of NADP+ to Gre2p in 100 mM HEPES Buffer.

ITC binding (BIND) experiments to determine the binding parameters of NDK (nitrononane-2,8-dione) to Gre2p in 100 mM KPi Buffer. Experiments failed due to very weak binding and poor solubility of NDK in buffer.

ITC binding (BIND) experiments to determine the binding parameters of HK ((5S,8S)-anti hydroxyketone) to Gre2p in 100 mM KPi Buffer. Experiments failed due to very weak binding and poor solubility of HK in buffer.

ITC multiple injection (MIM) experiments to determine the kinetic parameters of NDK (nitrononane-2,8-dione) and NADPH with Gre2p in 100 mM KPi buffer pH 7.5 Data from ITC recurrent single injection (rSIM) experiments were used to achieve proper fitting of kcat values.

ITC multiple injection (MIM) experiments to determine the kinetic parameters of NDK (nitrononane-2,8-dione) and NADPH with Gre2p in 1x PBS buffer pH 7.5 Data from ITC recurrent single injection (rSIM) experiments were used to achieve proper fitting of kcat values.

Badly chosen substrate concentrations for an intital rate experiment give data that makes it difficult to fit with a kinetic model and judge the quality of the fit.

Well chosen substrate concentrations for an intital rate experiment give data that makes it easy to fit with a kinetic model and judge the quality of the fit and use it as a basis for the final gold-round experiment.

Very well chosen substrate concentrations for an intital rate experiment give data that makes it very easy to fit with a kinetic model and judge the quality of the fit.

An initial choice of 5 widely spaced substrates enables a first, rough estimate of Km to inform the design of first-round experiments.

Raw data of the measurements obtained via DLS measurements. Can be opened with the Zetasizer software.

ITC recurrent single injection experiment for the reaction of NADPH and NDK with Gre2p in KPi, PBS and HEPES buffer (100, 1x, 100 mM, respectively, all at 25°C and pH 7.5). In this apj file, the data for the analysis of the first injection peak is stored. *.apj file

ITC recurrent single injection experiment for the reaction of NADPH and NDK with Gre2p in KPi, PBS and HEPES buffer (100, 1x, 100 mM, respectively, all at 25°C and pH 7.5). In this apj file, the data for the analysis of the second injection peak is stored. *.apj file

ITC recurrent single injection experiment for the reaction of NADPH and NDK with Gre2p in KPi, PBS and HEPES buffer (100, 1x, 100 mM, respectively, all at 25°C and pH 7.5). In this csv file, the data for the analysis of the both injection peaks is stored. *.csv file

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in 0.1%Tween-20-KPI buffer (100 mM, pH 7.5) at 25 °C. *.apj file

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in 0.1%Tween-20-KPI buffer (100 mM, pH 7.5) at 25 °C. *.csv file

Raw data of the measurements obtained via DLS measurements. Can be opened with the Zetasizer software.

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in HEPES buffer (100 mM, pH 7.5) at 25 °C. *.apj file

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in HEPES buffer (100 mM, pH 7.5) at 25 °C. *.csv file

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in KPI buffer (100 mM, pH 7.5) at 25 °C. *.apj file

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in KPI buffer (100 mM, pH 7.5) at 25 °C. *.csv file

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in PBS buffer (1x, pH 7.5) at 25 °C. *.apj file

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in PBS buffer (1x, pH 7.5) at 25 °C. *.csv file

ITC binding experiment for the binding of (5S,8S)-anti hydroxyketone (HK) to Gre2p. Note that binding was unsuccessful. *.apj file, experiment titrating 1 mM HK.

ITC binding experiment for the binding of (5S,8S)-anti hydroxyketone (HK) to Gre2p. Note that binding was unsuccessful. *.apj file, experiment titrating 200 mM HK.

ITC binding experiment for the binding of (5S,8S)-anti hydroxyketone (HK) to Gre2p. Note that binding was unsuccessful. *.csvfile, experiment titrating 1 mM HK.

This is the Jupyter Notebook to allow editing and working with the code. It is a simple example of how a Selwyn test can look like if it is passed (1.) or failed (2.).

This is the pdf of the Jupyter Notebook to allow looking at the notebook without installing anything. It is a simple example of how a Selwyn test can look like if it is passed (1.) or failed (2.).

This is the Jupyter Notebook to allow editing and working with the code. It is a simple example of how a progress curve experiment can look like.

This is the pdf of the Jupyter Notebook to allow looking at the notebook without installing anything. It is a simple example of how a progress curve experiment can look like.

This is the pdf of the Jupyter Notebook to allow looking at the notebook without installing anything.

NOTE THAT this pdf may be mangled when viewed on FAIRDOMHub, but should look fine when downloaded.

This is a zip file containing all the files and folders needed to analyse raw data, fit initial rates and create a "Michaelis-Menten plot". Instructions on usage are added in the notebook.

This is the Jupyter Notebook to allow editing and working with the code.

Design an initial rate experiment in four steps

This script aims to help you design high-quality initial rate experiments. It uses the Michaelis-Menten equation to simulate data for an enzyme with unknown enzyme reaction parameters. Four steps are needed:

1. Estimate the enzyme reaction parameters

Input: estimates of Km and vmax

and indicate the enzyme concentration concentration you are planning to use. If you have no ...

This is the pdf of the Jupyter Notebook to allow looking at the notebook without installing anything.

Design an initial rate experiment in four steps

This script aims to help you design high-quality initial rate experiments. It uses the Michaelis-Menten equation to simulate data for an enzyme with unknown enzyme reaction parameters. Four steps are needed: 1. Estimate the enzyme reaction parameters

Input: estimates of Km and vmax

and indicate the enzyme concentration concentration you are planning ...

Gre2p activity monitored by NADPH absorbance using different enzyme concentrations. Activity is measured after different storage conditions (treatments) and in presence of different amounts of tween.

A python workflow is used to analyse the data and create a plot where the outcomes of the Selwyn test are plotted. It requires the following directory structure:

./Sewlyn_test_forS19.py ./tween/M1.csv ./tween/M2.csv ./tween/M3.csv ./tween/M4.csv ./treatments/M1.csv ./treatments/M2.csv ./treatments/M3.csv ...

Gre2p activity monitored by NADPH absorbance using different enzyme concentrations. Activity is measured after different storage conditions (treatments) and in presence of different amounts of tween. Also analysis of DLS data of homogeneity of Gre2p samples measured before and after these treatments.

A python workflow is used to analyse the data and create a plot of the data. It requires the following directory structure:

./Script_for_S13andS18.py ./Script_for_S22.py ./Script_for_S15.py

and as ...

SOP describing how to perform Dynamic Light Scattering (DLS) measurements with Gre2p in cuvettes using a Zetasizer Nano DLS reader.

SOP describing how to perform an ITC multiple injections experiment with a MicroCal PEAQ ITC by Malvern.

SOP describing how to perform an ITC (recurrent) single injections experiment with a MicroCal PEAQ ITC by Malvern.

Python workflow for the analysis of ITC-BIND, ITC-MIM and ITC-(r)SIM experiments. Organized in a *.zip folder. Requires the following directory structure:

./ITC_analysis.py ./input/BINDING/.apj ./input/BINDING/.csv ./input/KINETICS/.apj ./input/KINETICS/.csv ./scripts/binding_neu.py ./scripts/kinetics_neu.py

And can be executed by running python ITC_analysis.py in the directory. Filenames for the input *.apj and *.csv files are defined in ITC_analysis.py. The output directory is written by ...

SOP describing how to perform an ITC binding experiment with a MicroCal PEAQ ITC by Malvern. Note: the DOIs from the Chemotion database for the synthesis of HK and NDK are missing in this version beacuse the DOIs do not exist yet (as of 27.04.21)

SOP describing the steps for use of the ITC device (MicroCal PEAQ ITC by Malvern).

SOP describing how to perform specific activity measurements with Gre2p in multiwell plates (96w) using an UV-Vis Plate reader (Synergy H1).

SOP describing how to perform activity measurements (inital rates) with Gre2p in multiwell plates (96w) using an UV-Vis Plate reader (Synergy H1).

Instructions and details on the data analysis workflow.

Master Bash script to launch Molecular Dynamics when running the Simulation part of Simulation Foundry. Make sure you followed "Preparations" instructions! Read the Manual!

Date recorded: September 14 2021

Enzymes are the catalysts of life, accelerating chemical reactions in living organisms. Enzyme reactions are widely applied in industry, for example as ingredients in laundry detergents, in the production of cheese, or to synthesize pharmaceutical drugs.

To apply enzymes in industrial processes, a detailed understanding of the molecular mechanisms is needed, requiring in depth studies on their reaction mechanisms. Such mechanistic studies can be performed with ...

dummy ifle fo rtesting file uplad and nbviewer

Scripts and Input Files for Simulation V1.5

Overview of the results obtained from the Simulation Foundry.

Overview of the quality control checks obtained from the Simulation Foundry.

This is a list of errors that can be produced if the Simulation Foundry is not used correctly - the messages are cryptic, hopefully the file is helpful to some.

Manual to Simulation Foundry Version 1.5.

Scripts and Input Files for Analysis V1.5

OPLSAA force field used by GROMACS on the BINAC Supercomputer (chem/gromacs/5.1.4-gnu-4.9, https://wiki.bwhpc.de/e/Category:BwForCluster_BinAC )

Screenshot of the directory structure required for the Simulation Foundry.