Enzyme immobilisation

In a normal digestive system all the food we eat is broken down this helps the body to absorb the food. Lactose is a natural sugar found in milk, it is a disaccharide carbohydrate (sugar) made up of two single sugar units (monomers) joined by a hydrolysis reaction (adding of water). The sugar lactose is not very sweet and is insoluble. The enzyme lactase produced in our body breaks lactose into its disaccharide forms-glucose and galactose. Glucose is the only sugar that can be absorbed into the bloodstream. In people that are lactose intolerant they do not have the enzyme to this job or the enzyme does not function well and therefore it could cause indigestion problems.

In this investigation I will immobilise enzymes by entrapping them in a gel. I feel there are many advantages of using the immobilised enzymes over the free ones, some advantages are:

* The enzymes can easily be separated from the mixture and so enzyme loss at the end of the experiment is minimised.

* Because the enzyme can easily be removed it is easier to re-cycle it, therefore it can be re-used a number of times.

* The reaction can be stopped by removing the enzyme.

* The immobilised enzyme has more stability than free ones and so o reaction rate is better as the enzyme is more firm.

I will immobilise the enzyme by

trapping them in an alginate gel.

Hypothesis-I predict that at the end of the experiment when I test the glucose strip, there should be a change in colour, and thus considerable amount of glucose should be present.

In the experiment the enzyme lactase will be the independent variable as this will determine the amount of conversion of lactose to glucose and galactose. Thus the glucose and galactose production will be dependant on this. I will measure the production at the end using a glucose test strip against a colour scale to see how much is present. Other variables such as the temperature I will keep constant so that they do not affect the investigation and results.

Apparatus- In order to carry out the experiment I will need to use various apparatus. Firstly I will need to make sure that I wear a laboratory coat to ensure safety and reduce the risk of any accidents. This is because the acids used could stain the clothes or hands.

I will need to get two 20 ml beaker. One will be needed to mix the alginate and lactase and the other will be needed at the end of the experiment when allowing the milk to drop. I will use a 20ml beaker because it is small and so it will allow me to make more accurate measurements. I will also need two 20cm measuring cylinder one to measure the calcium chloride and the other to collect the milk. I will use cylinders rather than as beaker as I feel it is easier to transfer the content.

I will then need to acquire 2cm lactase enzyme to convert the lactose into glucose and galactose. Also 8cm of alginate solution will be needed, to mix with the lactase to make the beads. In addition I will need 20cm of calcium chloride, to drop the beads into.

A glass rod will be needed to stir the alginate and lactose solution to ensure that the liquids are properly mixed.

I will then need to get three 10cm syringe. One will be needed to obtain the alginate and another one will be needed to get the lactase. Also I will need another syringe at the end of the experiment to place the beads into. I am going to use a syringe instead of a measuring cylinder or beaker, as I feel that this is smaller and so will allow me to measure more accurate definite measurements, which in turn will make the experiment fairer. Also I feel that a syringe is better as it is smaller measurements and so easier to handle

A boss clamp and a stand will be needed to hold the syringe over a beaker. I feel that using a clamp will be better than just holding the syringe over the beaker with my hands, as it more stable and the clamp will allow me to tighten the grip on the syringe. Also I will be able to control the pressing of the syringe better. A clip will ensure safety as the syringe will be firmly held so does not waver or fall.

Distilled water will be needed to wash the beads out after they have formed. I will use distilled rather than tap water because it does not have any chemicals added to it so I feel it is fairer.

A tea strainer will also be needed to strain the beads out. I feel a strainer needs to be used rather than just tipping the water out as it ensures that all the other calcium chloride is removed. Also it is safer as the beads do not come into contact with the hand.

I will then get a 220ml measuring cylinder to pour the milk in to. I will use a cylinder rather than just emptying the milk into the syringe as I could spill or pour too much in. I am going to use a small cylinder as the experiment is on a fairly small scale so I do not need large amounts.

A cut piece of nylon sheet will be required to place at the bottom of a syringe before transferring the beads into it, so that when the milk is poured in it does not flow out in large amounts. A Rubber tube will also be needed for this purpose. I will also use as hoffman clip to attach to the tubing so that I can adjust the clip to help me control the flow of milk.

At the end of the experiment I will need glucose strips to check the concentration of glucose produced /present in the solution.

Method- Before starting anything a laboratory coat must be worn. This will reduce the risk of any staining /harm of the liquids to the clothes and body. I will then wash my hands to ensure that they are clean and have no dirt on them.

Next I will obtain all equipment, so that I do not have to look for it in between the experiment and so that can carry the experiment out systematically. I will wash the beakers, cylinders and syringes and then wash/wipe them so that if any chemicals or substances are present they will be removed so as not to disrupt my experiment and results.

I will then get two syringes and collect the required amount of lactase and alginate. I will hold the syringe up to eye level and not tilt/slant it as the incorrect measurement may be obtained. I will also place a beaker on a flat surface when transferring from the syringes so that nothing is spilt.

Next I will mix the 2cm of lactase with the 8cm alginate gel solution into stirring gently with a mixing rod so that the solutions are mixed. I will then measure 20cm of calcium chloride in to a cylinder.

After the alginate solution is mixed I will collect it in a syringe filling it all up, so that I can test the maximum possible for the experiment. I will then get a clamp stand and clamp the syringe on with a boss and clamp, tightening as required. I will then place the chloride beaker under the syringe so that the solution can fall directly into it.

I will then press the syringe slowly releasing about 2cm solution each time in to the beaker. I will release the same each time so that I get the same sized beads, and so the reaction is fair. This is because bigger beads may cause longer for the lactose to be broken down. I will leave the solution in the calcium chloride for about 10 minutes to allow the beads to harden.

After 10 minutes are up I will strain the solution containing the beads into a tea strainer This is a better method than tilting the beaker as I can ensure all the liquid is out, so that no access calcium chloride remains. I will then wash the beads out with distilled water.

After I have washed the beads out I will get another syringe and cut a small piece of nylon gauze to be attached to the bottom of this. I will use another syringe as the previous one could still have some liquid which could contaminate the syringe. I will then attach a short length of rubber tubing to the end of the syringe and screw a Hoffman clip on to it. The hoffman clip will be needed to control the flow of milk.

I will remove the part of the syringe that is pressed so that the syringe is open from the top. I will then transfer the beads in to the syringe. Next I will attach a rubber tube to the end of the syringe and fasten a hoffman clip to the tube to control the flow of the milk. I will then clamp the syringe over a clean beaker, tightening and adjusting with the clamp to get the syringe stable.

I will then pour some milk into a cylinder as it could all spill if I transferred it straight from the milk bottle to the syringe. I will then transfer a little in to the syringe. After a while I will unscrew the Hoffman clip slowly to allow drops to fall into the beaker.

Finally I will get a glucose test strip and dip it in to the milk. I will then wait a few seconds to allow the glucose to absorb on to it and then read the % of glucose present in the lactase treated milk against a colour scale. I will then wash my hands to remove any substances that may have come into contact with my hands.

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