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. The Effect of Temperature on the Activity of Proteolytic Enzymes

Principle

The ability of enzymes to accelerate biochemical reactions in mild conditions opens up many opportunities for improving large scale industrial processes. For example, Wheat and Maize starches are now converted to glucose and fructose syrups without the need for inorganic acids. This is achieved by using naturally produced enzymes from renewable resources. Micro organisms, including bacteria, yeasts and filamentous fungi produce a vast array of enzymes capable of breaking down most foodstuffs and some of the most toxic chemicals produced by man eg, PCB’s and organophosphorus compounds [insecticides and sheep dip].

The micro organisms which are grown in fermenters [capacity 10,000L] are selected for their ability to produce high concentrations of the desired enzyme. Sometimes this will involve the use of genetically modified bacteria or fungi.

In this practical, proteolytic enzymes are tested for their ability to breakdown the protein gelatin at different temperatures. The commercial application of this is in the manufacture of biological washing powders. As well as attacking protein stains directly, the enzymes degrade the protein glue which sticks the stains onto the fabric, enabling the detergent to lift them free.

The first enzyme detergents were marketed in 1914 and contained washing soda [Na carbonate] and small amounts of pancreatic juice containing proteolytic enzymes. The very alkaline conditions [pH 10¬-11] reduced the activity and stability of the added enzymes and the product was not a success. By 1961, a Dutch Company Novo had produced a bacterial protease from Bacillus licheniformis which remained active and stable in alkaline washing conditions and the enzyme was given the trade name ALCALASE. It is capable of working at very high temperatures eg 85oC. Since then a whole battery of enzymes have been added capable of degrading fat and causing softening of cellulose fibres in clothes. Following the introduction of optical brighteners in the 1950’s the main development has been to produce low temperature detergents especially for the European market. Western Europe is the only region where by tradition, hot washes are considered essential for cleaning clothes. In USA and Canada, water enters the machine directly from the hot water supply and washes are usually done at 55oC. Proteases work best at 55 60oC and so the search is on for enzymes which function even better between 20 36oC.

Method

You are provided with samples of two proteolytic enzymes, 0.1% Alcalase and 0.1 % Savinase. Label 4 Boiling tubes for Savinase and the corresponding incubation temperatures of 20o C [room temperature], 33oC, 40o C and 60o C.

Pipette 10.0ml of Savinase into each Boiling tube and place in the appropriate temperature water bath for 10 min to equilibrate.

Take a further 10.0ml of Savinase and boil in a boiling water bath for 5min. Allow the sample to cool and then place at 60oC. This should act as a control since boiling denatures the enzymes irreversibly.

Repeat the procedure using 0. 1% Alcalase.

Using a 5% solution of a commercial washing powder [BRAND X!!] incubate at two temperatures only, room temperature and 60o C.

To start the reaction, add a small piece of photographic film with an observable image to each tube. Start the stop clock and determine the time taken for the images to be destroyed at each incubation temperature.

THINK !!! – How will you determine the end point of this reaction ? It is suggested that you take the final loss of the gelatine from the film base as your end point.

You should inspect the tubes every 5 min – you will need to gently shake the tube each time to determine the extent of degradation. Remember to shake all tubes in the same way and for the same duration of time.

At 20o C [room temperature], the experiment will need to set up for the duration of the practical ie 1.5 hours before any changes will be seen.

Results

Savinase

Temp oC Time (Mins) to image disappearance
20 40 mins 50 secs
33 28 mins 20 secs
40 15 mins 40 secs
60 25 mins 30 secs

Alcalase

Temp oC Time (Mins) to image disappearance
20 12 mins 40 secs
33 8 min 10 secs
40 6 mins 15 secs
60 4 mins 20 secs

Brand X

Temp oC Time (Mins) to image disappearance
60 RT 40 mins 60oc 13 mins 20 secs

1. From the results what would be your recommendations with respect to the inclusion of Savinase and Alcalase for hot wash and cool wash detergents?

2. For which wash temperature was commercial washing powder (Brand X) designed? What would your expectations be (ie what is the effect on the enzymes) if the detergent was not used at this temperature?

Any help would be great .

Thanks