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Team:UC Davis/Protein Engineering Test
From 2014.igem.org
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<b>Enzyme, Substrates, and Coenzymes</b> | <b>Enzyme, Substrates, and Coenzymes</b> | ||
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| - | The main components of our assay are aldehyde dehydrogenase (ALDH), the NAD+ coenzyme, and the aldehyde substrate. Since NADH is a product of oxidation by aldehyde dehydrogenases, we were able to use a simple spectrophotometric approach to quantify the reaction rate of these enzymes on different aldehyde substrates. Using a plate reader, we tracked the change in absorbance at 340nm and measured the accumulation of NADH over time as aldehydes were converted into carboxylic acids. In addition to these basic components, there were several necessary additions made to our assay. | + | The main components of our assay are aldehyde dehydrogenase (ALDH), the NAD<sup>+</sup> coenzyme, and the aldehyde substrate. Since NADH is a product of oxidation by aldehyde dehydrogenases, we were able to use a simple spectrophotometric approach to quantify the reaction rate of these enzymes on different aldehyde substrates. Using a plate reader, we tracked the change in absorbance at 340nm and measured the accumulation of NADH over time as aldehydes were converted into carboxylic acids. In addition to these basic components, there were several necessary additions made to our assay. |
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| - | <td>NAD+</td> | + | <td>NAD<sup>+</sup></td> |
<td>4mM</td> | <td>4mM</td> | ||
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Revision as of 01:48, 18 October 2014
Assay Components
Enzyme, Substrates, and Coenzymes
The main components of our assay are aldehyde dehydrogenase (ALDH), the NAD+ coenzyme, and the aldehyde substrate. Since NADH is a product of oxidation by aldehyde dehydrogenases, we were able to use a simple spectrophotometric approach to quantify the reaction rate of these enzymes on different aldehyde substrates. Using a plate reader, we tracked the change in absorbance at 340nm and measured the accumulation of NADH over time as aldehydes were converted into carboxylic acids. In addition to these basic components, there were several necessary additions made to our assay.
| Standard NAD(H) Assay Stock Solutions | |||
|---|---|---|---|
| Stock | Volume in Assay | Components | Concentration |
| Base | 130uL | Tween 20 | 1.54% (v/v) |
| DTT | 1.11 mM | ||
| Substrate | 20uL | Aldehydes | 10x Final Concentration in Isopropyl Alcohol |
| Enzyme | 50uL | ALDH | 0.004 mg/mL |
| NAD+ | 4mM | ||
| DTT | 1.11mM | ||
| 200uL | Final Assay Volume | ||
| Order of addition into plate to initiate assay: substrate, aldehyde, enzyme. Assays were conducted in a Costar 96 well flat bottom plate. Biotek Epoch/Synergy H1 plate readers were used to measure NADH accumulation at A340 over the course of 30 minutes. Unless stated, all solutions were prepared in 11.1mM Potassium Phosphate, 111.1mM Potassium Chloride solution. | |||
Isopropyl Alcohol
We were faced with two options when it came to introducing the aldehydes from an olive oil sample to our enzymes: solubilization or extraction. We determined that the extraction of aldehydes from a sample of olive oil would be ideal for our application; this would avoid the use of emulsifiers and other compounds which could potentially interfere with the enzymes and electrode. We used isopropyl alcohol as a representative solvent to extract the aldehydes from a sample of olive oil. Isopropyl alcohol was present at a concentration of 10% during all of our assays.
Dithiothreitol (DTT)
Aldehyde dehydrogenases possess a catalytic cysteine group which attacks the carbonyl carbon of an aldehyde to create an intermediate thiol ester. Since several cysteine groups exist around the catalytic center of the aldehyde dehydrogenases we investigated, it was imperative that these residues did not oxidize to produce disulfide bridges. Thus, 1mM dithiothreitol (DTT) was used in all assays to prevent oxidation of the enzyme.
Tween 20
Aliphatic, straight-chain aldehydes are inherently hydrophobic and had a difficult time solubilizing in our initial assay conditions. Even in 10% isopropyl alcohol, longer chain aldehydes (e.g. nonanal, decanal) started to crash out of solution at concentrations greater than 100uM. To alleviate these issues, we conducted our assays in a final concentration of 1% Tween 20. While we found that the electrode could not operate in the presence of Tween 20, it was necessary in our enzyme characterization assays to collect data for long chain aldehydes.
Initial Screening, Selection, and Characterization
Screening
With our enzymes expressed and a robust assay ready to go, we needed to identify aldehyde dehydrogenase enzymes with unique specificity profiles. Recall in the context of our device that we needed enzymes which would selectively oxidize aldehydes in a particular category (medium saturated, long saturated, unsaturated) to produce NADH. NADH may then be quantified using either spectrophotometric or electrochemical methods to determine the original concentration of a particular category of aldehydes in a sample.
We screened all 24 aldehyde dehydrogenases (two mutants did not express) against all sixteen aldehyde substrates we previously identified to occur in olive oil. We assayed each enzyme against 100uM aldehyde substrate and calculated the steady-state velocity for each enzyme-substrate pair. This panel of 16 steady state velocities served as a fingerprint for the specificity profile of each enzyme. We found three aldehyde dehydrogenases with unique substrate specificity profiles: WT Escherichia coli ALDH, WT Rattus norvegicus ALDH, and W176Q Escherichia coli ALDH.
Click here to see the screening data!
Michaelis-Menten
Now that we had identified three aldehyde dehydrogenases with unique specificity profiles, we proceeded to experimentally determine the kinetic constants (kcat and KM) of these enzymes on each substrate in our assay conditions.
Click here to see the experimentally derived Michaelis-Menten constants for our three enzymes!
Aldehyde Extraction from Olive Oil
Since olive oil is not miscible in our assay conditions, we needed to extract the aldehydes from our olive oil samples to assay them against our aldehyde dehydrogenases. We devised a simple protocol for the extraction of aldehydes from one sample of olive oil:
Mix olive oil and isopropyl alcohol in a 1:1 ratio in an eppendorf tube and vortex for at least 30 seconds.
Centrifuge the olive oil/isopropyl alcohol emulsion for at least 5 minutes at 14,800 rpm to separate the phases. The isopropyl alcohol with the extracted aldehydes will sit on top of the olive oil in the eppendorf tube. Pipet off the isopropyl alcohol into a new eppendorf tube, the olive oil can be disposed.
Mix the extracted aldehydes with the stock base buffer (see assay components) in a 2:13 ratio (add at least 20uL of extraced aldehydes in isopropyl alcohol to 130uL of stock base buffer). Aliquot 150uL of this mixture into a Costar 96 well flat bottom plate.
To initiate the assay, add 50uL of the enzyme stock solution (see assay components). Measure the accumulation of NADH at A340 over the course of 30 minutes.
As the OD vs. Time graph shows below, we are able to produce a clear signal from aldehydes extracted from olive oil (as compared to an extraction on refined canola oil lacking aldehydes).
