In this task I will investigate how the enzyme lactase breaks down the disaccharide lactose into monosaccharides, which are glucose and galactose. The main aim will be to get lactose – free milk as milk contains lactose. When we drink milk we need lactase (enzyme) to break down the lactose, which is contained by the milk. Once I have created my own method of enzyme immobilisation of lactose using lactase I will then construct a bioreactor.
I have looked at and researched on the Internet and found many ways (methods) to carry out this experiment. I have also noticed that some are long and complicated to understand and follow but I have managed to get one decent method as a basis for my own method to be created. These have been attached at the appendix.
I will carry out this experiment using 5 different temperature ranges, as this will give me good, accurate and reliable results. It will give me more data of results to compare and analyse. So the 5 temperature ranges that I have chosen are: 30°C, 35°C, 40°C, 45°C and 50°C (independent variables). I will also repeat the experiment 3 times for each temperature to get more reliable results. I will make sure the time the mixtures are kept in the water baths are temperature equilibrium, which means that they will be kept in the water baths until they reach that temperature. This will require me to use a thermometer and place it in with the mixtures (5mins or more).
2cm3 lactase enzyme
8cm3 Sodium alginate solution
100cm3 Calcium chloride solution
Hot water baths set at 30°C, 35°C, 40°C, 45°C and 50°C. •
Small beakers x 4
Syringes 5cm3 (precision +/- 0.05cm3)
Glucose strips/reagent strips Semi-quantitative (Diabur 5000)(max 3%) •
Test tubes and rack
Plastic tea strainer
Distilled water 100cm3
Pasteurised milk (semi-skimmed)
Stop watch (+/- 0.05sec)
First get all the apparatus out and set up.
Mix 8cm3 sodium alginate solution with 2cm3 lactase enzyme solution in a beaker, and transfer into a syringe.
Add this mixture drop-wise into the calcium chloride solution (100cm3). Alginate beads will form in the beaker, leave for 15 to 20 mins for the beads to harden.
Strain off the beads using the tea strainer and rinse with distilled water.
Transfer the beads into a test tube.
Place the test tube containing the beads and a test tube containing 2cm3 of milk into a water bath of 30°C and wait until they have reached the temperature of the water bath. (About 5mins).
Then pour the milk in the test tube into the test tube with the beads and start the stopwatch immediately.
After 5mins test the solution with a glucose strip and record the amount of glucose present.
Repeat this for each temperature 3 times.
Repeat this for 35°C, 40°C, 45°C and 50°C.
Note: the alginate beads can be re-used after been rinsed with distilled water.
Then this experiment method can also be used for the mobilised free enzyme. This can be done by simply repeating the above method but without adding calcium chloride which means that steps 1, 2 and 3 don’t need to be taken.
Reference: Michael Roberts 2001
A simple batch bioreactor to be used by both immobilised and mobilised enzyme (lactase)
Building a coke Bottle Bioreactor
Coke bottle bioreactors are designed to be used as tools for composting research. They are small and inexpensive enough to enable students to design and carry out individualized research projects, comparing variables such as reactor design, moisture content, and nutrient ratios of mixtures to be composted. This is simply a batch bioreactor, which means that it is not circulated.
Two 2-litre or 3-litre coke bottles
One smaller container, about 5-cm high, that fits inside the...
References: • http://www.css.cornell.edu/compost/soda.html
• Biology book: Tim Kino, Micheal Reiss, Micheal Roberts 2001
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