Watch an oxidation reaction turn green
Use a validated ABTS assay to compare colour formation with and without laccase. This creates a visible analytical reaction without promising that any household dye is a suitable substrate.
Use these original S&T teaching drafts after a teacher or technician has piloted the exact product, substrate and assay. Recipes and timing targets are proposed starting conditions, not validated performance claims. Review current SDS and institutional guidance; wear eye protection, avoid enzyme aerosols and splashes, and keep powders under technician control. No tasting. Keep controls at the same final volume, pH, temperature and stirring conditions. Plan reagent-specific waste disposal.
Laccase in nature
Laccases are copper-containing oxidases found in fungi, plants and some other organisms. Fungal laccases can participate in transformations of aromatic compounds associated with plant materials. They transfer electrons from suitable substrates to oxygen, which is reduced to water.
What chemical reaction does it catalyse?
Enzymes speed up a reaction without being consumed in the overall process. The preparation, accessible substrate and reaction conditions determine what you can measure.
Industrial applications
Laccases are investigated and used for selected textile, pulp, beverage and environmental biocatalysis processes; performance depends strongly on substrate and conditions.
Experiment overview
Use a validated ABTS assay to compare colour formation with and without laccase. This creates a visible analytical reaction without promising that any household dye is a suitable substrate.
Equipment and materials per working group
- Technician-prepared ABTS working substrate, for example 0.5 mM in compatible acetate buffer near pH 5
- Technician-prepared laccase working solution, initially 0.01% w/v
- Three clear cuvettes or tubes, pipettes and timer
- 40°C bath if the selected validated method supports it; optional 420 nm colorimeter
Plan a class of 30
Use 10 working groups of three. Multiply the per-group quantities in this guide by 10, then allow an appropriate preparation reserve. Keep concentrations unchanged when scaling. Assign a timer, a practical operator and a recorder, then rotate roles. Set up teacher-prepared comparison samples in advance where a reaction takes longer than the lesson.
Teacher and technician preparation
- Review the SDS for ABTS and the enzyme. Pupils receive prepared liquids; no handling of dry powders.
- The 0.5 mM ABTS / 0.01% enzyme starting point is a teaching pilot formulation, not a validated S&T protocol. Confirm compatibility with the current laccase preparation and selected reference method.
- Pilot 2.9 mL substrate plus 0.1 mL enzyme working solution. Dilute enzyme if colour is immediate or saturating; run substrate and enzyme blanks.
Use enough working enzyme to make a clear comparison in the intended time window. Do not automatically add more concentrate: reactions can finish before measurement, stocks can alter pH or background colour, and some substrates remain slow even at a high dose. Pilot the proposed recipe, then record the final preparation and dilution on the results sheet.
Step-by-step class method
- Label ENZYME, CONTROL and ENZYME BLANK.
- Add 2.9 mL selected ABTS substrate to ENZYME and CONTROL and 2.9 mL buffer to the blank.
- Equilibrate all tubes at the chosen method temperature.
- Add 0.1 mL selected laccase working solution to ENZYME and the blank; add 0.1 mL matching diluent to CONTROL.
- Mix gently and start timing.
- Record visible colour at 0, 1, 3, 5 and 10 minutes, or record absorbance at 420 nm if using a compatible method.
- Compare the response to both blanks and identify a useful unsaturated interval.
- Discuss the roles of substrate specificity, oxygen and electron transfer.
What should students look for?
A compatible ABTS assay develops a blue-green/green signal; its speed and intensity depend on the preparation and conditions.
Interpretation and troubleshooting
ABTS chemistry is an advanced analytical demonstration, not a food experiment. Do not generalise this response to all dyes or claim that a colour change establishes pollutant removal.
Questions for the class
- What changed in the enzyme-treated sample compared with the control?
- What bond or substrate does this enzyme act on?
- Which conditions did we keep the same, and why?
- Could colour, pH, warming, stirring or background sugars explain part of the result?
- What would you measure or change in a follow-up investigation?
Your investigation sheet
Name: ____________________ Group: ______ Date: __________
Prediction: _______________________________________________________
Enzyme preparation / batch: ________________________________________
Final working dilution and dose: ____________________________________
Substrate: _______________ pH: ______ Temperature: ______ °C
| Time / min | Enzyme + substrate | No-enzyme control | Enzyme-only blank / notes |
|---|---|---|---|
| 0 | ________________ | ________________ | ________________ |
| 2 | ________________ | ________________ | ________________ |
| 5 | ________________ | ________________ | ________________ |
| 10 | ________________ | ________________ | ________________ |
| Later: ___ | ________________ | ________________ | ________________ |
Conclusion supported by our control: _________________________________
One limitation and one improvement: __________________________________
Background sources and product details
This is an original teaching adaptation. External methods establish useful principles and assay formats; they do not validate the suggested doses for S&T preparations.