Enzyme preparation for experiments.

Enzyme preparation for experiments: I would like the correct preparation of these enzyme solutions: pepsin, trypsin and amylase.

Thanks.

Voting: 
0
No votes yet
Publication Date: 07 May 2015
Asked By: Dianne
Showing 1-1 of 1 Responses

Enzyme preparation for experiments.

Expert Answer

Firstly, please read and be familiar with the “General Safety Precautions when using Enzymes” below prior to preparing enzyme solutions. Enzymes in powder form are considered hazardous substances1,2,3. However, in dilute aqueous concentrations, they are considered to be a low hazard.

It is important to understand that enzymes, when dissolved into solution, are much less stable than in powder form and lose their activity quickly. Therefore, it is best to prepare only what is required, and only just before use. Enzymes in powder form should be stored in the fridge (4° C) unless otherwise specified. Diluted solutions can be stored in the fridge, but should be used within of an hour or two of preparation and be kept on ice during an experiment.

Enzymes are usually made up as a percentage concentration. A 0.5% to 1% w/v solution is generally suitable for enzyme digestion practicals carried out in schools. It is always best to use the lowest concentration and smallest amount possible. The optimum reaction conditions can be different for each enzyme. It should also be noted that both trypsin and amylase work optimally around a neutral pH, whilst pepsin requires a pH of 1.5–2 to be active. The protocol for the procedure that you are following should indicate the type and amount of acid required to acidify a reaction using pepsin. It may mean that an acid, in many cases hydrochloric acid, is added to the reaction, or that pepsin is made up in a dilute acidic solution instead of water.

Here is a recipe that is suitable to prepare 100 mL of a 0.5% w/v solution of trypsin, pepsin or amylase using distilled water.

Wear PPE: safety glasses, gloves, laboratory coat, face mask or work in a fume cupboard that is not turned on to minimise exposure to dust or aerosols. If working outside a fume cupboard, make sure you are in a draft-free area.

  • Weigh out 0.5g of the enzyme.
  • Add to 80mL of distilled water at room temperature in a beaker.
  • Stir gently to dissolve.
  • Adjust to a final volume of 100ml.
  • Store at 4° C (fridge) for a short period of time or on ice during use.
  • Do not heat or allow the solution to froth, as this will denature the enzyme.

Working with Enzymes:

Enzymes are proteins that are catalysts of chemical reactions. Catalysts increase the speed of the chemical reaction but do not form part of the final product. Enzymes act on substrates to make products in a chemical reaction and they are highly specific to the reactions they catalyse (the lock and key model).

It is always advisable to check the enzyme reaction is working as required and make adjustments to the conditions and concentrations if needed before any practical class. Enzyme activity is affected by concentration, temperature, pH, substrate concentration and can be affected by the age of the reagents.

Digestive Enzymes:

Amylase is found in saliva in the mouth (salivary amylase) and in the pancreatic juice in the pancreas (pancreatic amylase). It is an enzyme that breaks down starch into sugar. Amylase operates optimally at a pH of 6.7 to 7.0 and at 37° C.

Pepsin is the main gastric enzyme that digests proteins into their component peptides and amino acids. Pepsin is secreted in the stomach and operates optimally in an acidic environment around pH 1.5—2.

Trypsin, which is secreted in the small intestine, digests proteins into their component peptides and amino acids. Trypsin operates optimally at a neutral or slightly alkaline environment of pH 7–9.

General Safety Precautions when using Enzymes:

Safe handling of enzyme preparations can be accomplished through proper work practices, engineering controls, and use of personal protective equipment.

Note: Enzymes are biologically active proteins. It is advised to avoid inhalation of enzyme dust or aerosols, which can lead to sensitisation and allergic reactions. Enzymes may cause asthma and are irritating to the eyes, respiratory system, mucous membranes and skin. Always wear safety glasses and gloves. When working with powdered enzymes, wear a dust mask or work in a fume cupboard, that is not turned on, to minimise exposure to any dust. Always use practices that do not generate dust or aerosols.

Enzymes in powder form are hazardous substances1,2,3. However, in dilute aqueous concentrations, they are considered to be a low hazard.

Minor spills should be cleaned up immediately, without generating dust. Place waste into a labelled container for disposal via a waste contractor.  Do not discharge waste into the sewer or waterways.

Science ASSIST recommends you conduct a site-specific risk assessment to assess and control the risks. You will need to determine how to safely prepare, handle and dispose of the solution. We have developed a Risk Assessment template for schools to use, see Risk Assessment Template.

References

Ball, Peter J ‘Working with Enzymes’ Southern Biological website http://file.southernbiological.com/Assets/WorkshopNotes/WorkshopNotes_Enzymes.pdf (Accessed May 2015)

‘Enzyme action – Amylase (clarase)’, Southern Biological website http://file.southernbiological.com/Assets/Products/Chemicals/Enzymes/MC23_31Amylase_(clarase)/AmylaseClarase(MC23_31)InfoSheet.pdf (Accessed May 2015)

‘Enzyme action – Amylase (diastase)’, Southern Biological website http://file.southernbiological.com/Assets/Products/Chemicals/Enzymes/MC23_35PAmylase_(diastase)/AmylaseDiastase(MC23_35P)InfoSheet.pdf (Accessed May 2015)

‘Enzyme action – Pepsin’, Southern Biological website http://file.southernbiological.com/Assets/Products/Chemicals/Enzymes/MC23_2MPepsin/PepsinInfoSheetMC23_2M.pdf (Accessed May 2015)

‘Enzyme action – Trypsin’, Southern Biological website http://file.southernbiological.com/Assets/Products/Chemicals/Enzymes/MC23_1M-Trypsin/TrypsinInfoSheet.pdf (Accessed May 2015)

‘Enzymes’ Royal Society of Chemistry website http://www.rsc.org/Education/Teachers/Resources/cfb/enzymes.htm (Accessed May 2015)


1 Chemwatch, November 2011.Material Safety Data Sheet: Trypsin

2 Chemwatch, May 2013.Material Safety Data Sheet: Pepsin

3 Chemwatch, April 2011.Material Safety Data Sheet: Amylase

Thank you for submitting an answer to this question. Your response has been sent to our administration team for moderation.