In a jam and out of juice
Safety and using enzymes
Orange and apple tied together

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SAFETY GUIDELINES FOR HANDLING ENZYMES

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The enzymes suggested for work in this booklet are safe to use, provided they are handled appropriately. While all the enzymes are food-grade products, items made with them should not be consumed. There are two reasons for this. Firstly, the proportions of enzyme products used in the activities suggested here are far greater than are normally used in the food industry. This is so that investigations can be conducted within the time of a normal practical session. Secondly, the enzymes have not been handled aseptically, so they (and the products made using them) may have been contaminated.

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USING ENZYMES

Please note: This is general advice from the NCBE for the use of enzymes in school investigations. It is not intended as a guide to industrial or research practice.

Safety guidelines for practical work with enzymes in schools
can be found here.

Enzyme activity units

The internationally-recognised unit of enzyme activity is the Katal (abbreviated to kat). It is defined as the enzyme activity which transforms 1 mole of substrate per second under optimal conditions.

In practice, few enzyme suppliers use the Katal, preferring instead units that reflect the preferences of their typical customers. For example, bakers are familiar with amylase activity expressed in 'SKB units', whereas brewers traditionally refer to amylase activity (which they call diastatic activity) in 'Degrees Lintner'. To obtain these various measures of enzyme activity, different assay methods have been used, and unfortunately there is no means of comparing dissimilar tests without conducting further practical work.

 

Planning your own enzyme tests

A solution to this problem is to develop your own tests. This is not as difficult or time-consuming as it might seem, and will almost certainly save you time (and money) in the long run.

  • Step 1: Define your operating conditions
    Identify the pH and temperature likely to occur in your proposed investigation. For example, a simple starch digestion experiment might use a neutral starch suspension in a test tube at room temperature. Decide how you are going to monitor the course of the reaction. It maybe a colour change (like starch and iodine solution); or it may depend upon chemical tests such as glucose test strips. Specify how long you wish the reaction to take. Sometimes it is useful if a reaction can occur quickly so that it fits comfortably within a practical session. Alternatively, you may want the reaction to proceed relatively slowly so that samples can be taken at convenient intervals to follow its course.
  • Step 2: Consult the enzyme data sheet
    Study the data sheet supplied with the enzyme. The methods used to measure the enzyme's activity mentioned there may be similar to your proposed practical.
    e.g., the data sheet supplied with Novozymes Celluclast states the amount of enzyme and the conditions needed to break down waste paper to fermentable sugars (e.g., you might be trying to produce glucose from wastepaper). Study at the graphs describing the enzyme's activity and stability. Very often the temperature and pH at which a particular enzyme works best are not those at which it is most stable. In practice this means that, for example, you may have to use a protease at less than its optimum temperature or pH (and therefore sacrifice speed) to ensure that it remains stable over the period of the investigation. Or a small degree of hydrolysis may be achieved at a high temperature before the enzyme is inactivated. Should either of these approaches prove unsatisfactory, the enzyme dosage can be increased.
  • Step 3: Produce a plan based on your requirements and information from the data sheet
    To start with, it is often easier to use a relatively high enzyme concentration at sub-optimal pH and temperature, then to reduce the amount of enzyme once you know that the reaction is going to proceed within the time you have available.
  • Step 4: Conduct the practical tests
    If 'bucket chemistry' doesn't work, or the results of the reaction aren't very obvious, you may need to modify your procedure.

 

Pushing enzymes to their limits

Sometimes it is not possible to achieve an optimum environment for enzyme activity in the classroom. Perhaps a water bath is not available, or it is difficult to accurately adjust the pH of the substrate. However, enzymes can and do operate under non-ideal conditions, and it may be useful to bear the following points in mind:

Sometimes lowering the temperature will broaden the effective pH range of an enzyme. e.g., Novozymes Lactozym will hydrolyse the lactose in acidic whey from home made soft cheese, at room temperature, even though the enzyme's optimum pH is around 7.0.

Consider high temperature/rapid processing. The Q1O rule states that for every 10 C rise in temperature, the enzyme will react twice as fast. Of course, this is only true up to a point i.e., until the enzyme is denatured, but substantial catalysis can still be achieved in a short time if lesson timing demands it e.g., washing powder proteases can clear particular types of photographic film in just five minutes at 65 C.

Increase the substrate concentration. High levels of substrates tend to stabilise enzymes, even under non-ideal conditions. The enzyme optimum and stability curves on Novozymes data sheets are often obtained from reactions where the substrate concentration is 10% w/w or less. If you use substantially greater levels of substrate, you may be able to raise the temperature or reduce the enzyme dose. Note: Some enzymes, such as invertase, are inhibited by high substrate concentrations.

This advice on using enzymes in schools, by Dean Madden, first appeared in the NCBE Newsletter in Summer 1991.

SPECIAL ENZYME PACK

The NCBE supplies the individual enzymes required for the practical work described in this booklet. The Centre also sells, at a substantial discount, a pack containing all of the enzymes (five types in total) together with a printed copy of 'In a jam and out of juice'.

For further information, please see the 'MATERIALS' section of the NCBE Web site.

Copyright National Centre for Biotechnology Education, 2006 | www.ncbe.reading.ac.uk