The Effect of Temperature on Lactase Immobilised Enzymes in an Enzyme Controlled Reaction

The Effect of Temperature on Lactase Immobilised Enzymes in an Enzyme Controlled Reaction
Aim: To investigate how a change in temperature will affect the rate at which lactase enzyme will hydrolyse lactose disaccharide sugar into glucose and galactose monosaccharide sugars. Background: Enzymes are biological catalysts which speed up chemical reactions but do not get involved in the chemical reaction, they remain unchanged. Enzymes are large and complex molecules that are synthesised by the cell to perform a specific function. These biological catalysts are very important because they speed up the rate of reaction at which they catalyse, otherwise the chemical reactions that take place in all living organisms would be too slow to support them. Enzymes are specific, they only catalyse one substrate. Enzymes have a three-dimensional shape due to the way the amino acid chain that makes up the chain is folded.
This is the tertiary protein structure. Several amino acid groups on the surface on the enzyme fold inwards forming the active site, which is complimentary to a specific substrate. This affinity between the active site and substrate molecule causes them to join (forming enzyme-substrate complex), the reaction takes place and then the products are released, the enzyme is free to take part in another reaction. Temperature is one factor which affects enzymes. The higher the temperature the faster the rate of reaction as per kinetic theory (the molecules gain more energy so they move faster, causing more collisions the enzymes and substrate molecules, thus forming more product). But if the temperature rises above a certain point the rate of reaction decrease, because the enzyme has started to denature, the enzyme shape is changing affecting the affinity between the enzyme and substrate molecule. The graph below shows the effect of temperature on enzyme activity: Temperature and the rate Other factors which affect enzymes are:
Substrate concentration ? As the substrate concentration
increases, their reaction rate initially increases proportionately
as collisions between enzyme molecules and reactants become more
frequent. When the enzymes begin to approach the maximum rate at
which they can combine with reactants and release products,
further increases in substrate concentration have no effect on
there reaction rate.
Inhibitors ? enzymes are susceptible to inhibition by molecules
that resemble a substrate molecule. The inhibitor binds with the
active site blocking access for the normal substrate, resulting in
the decrease in rate of reaction. These are called competitive
inhibitors as they compete with the substrate for the active site.
Non-competitive inhibitors, these inhibitors do not reduce
asseccibility to the active site but cause changes in the folding
structure on the enzyme thus reducing enzyme efficiency. Such
inhibitors are poisons and toxins (e.g. cyanide and carbon
pH ? Each enzyme has an optimal pH range that help maintain its
normal configuration. Most enzymes have an optimum pH of 7-8, but
there are some that prefer the extreme pH levels (e.g. pepsin
enzymes prefer pH of 2 as they work in the stomach. The tertiary
structure of a protein depends on interactions such as hydrogen
bonding, between R groups. A change in pH can alter the ionization
of these side chains and disrupt the normal configuration and in
some cases denature the enzyme.
Lactase is an enzyme found in the small intestine that breaks down lactose (naturally occurring sugar in milk) into galactose and glucose. 70% of the adult population are lactose-intolerant, i.e. they have difficulty in digesting lactose, this is because they do not produce sufficient amounts of lactase. Lactose is a disaccharide with one glucose sugar molecule bound to one galactose sugar molecule. Once lactose is split, our bodies readily metabolize the glucose and galactose products. Lactose is found is dairy products and is ingested nearly every day, and so lactase enzymes are needed to break the lactose down. An alternative way of drinking milk is to drink lactose-free milk (milk with lactose already broken down) which is done by using enzyme immobilisation. Enzyme immobilisation is when enzymes are attached/held in an insoluble material but the enzymes are still active. Advantages include: 1. enzymes can be re-used 2. enzymes can be used continuously 3. enzymes become more stable when immobilised 4. enzymes can be added or removed easily to control reaction 5. Enzymes are easily separated from the products of the reaction Prediction: I predict that as you increase the temperature the rate of reaction will increase until it reaches a certain temperature (anything over 40C, so around 50-60C, it would normally be 40C as that is same temperature of human body but as enzymes is now immobilised it is more stable so it can withstand higher temperatures). Method: I transferred 10cm³ of 2% sodium alginate solution into a small beaker. I used a different syringe to transfer 2cm³ of 2% lactase enzyme into the same beaker and mixed it thoroughly. I withdrew this mixture with a syringe. Gently I added this mixture drop by drop to 1.5% calcium chloride solution in a large beaker. I held the mouth of the syringe about 5cm above the calcium chloride. The calcium chloride acts as an hardening agent so I allowed the beads to harden for 5 minutes. I used a strainer to rinse the beads in water and transfer to a test tube. Once I placed a clinistix into the test tube I added 5cm³ of milk and then started timing. I then recorded the time taken for a colour change upon the clinistix. After this I rinsed the beads in water and repeated the above with the other 5 temperatures using a glass beaker for a water bath. Results: A results table to show the time taken for the lactase enzyme to hydrolyse the lactose sugar at different temperatures. Temperature (ºC) Time taken for clinistix to Rate of reaction change colour (in seconds) (100/sec) 20 275 0.36 30 180 0.56 40 120 0.83 50 85 1.18 60 60 1.67 Rate of reaction worked out by dividing 100 by the time taken for the clinistix to change colour. I worked out rate of reaction as then it can be plotted on a graph so then I can easily see the effect of temperature on the effiency of the immobilised lactase enzyme to break down the lactose. Analysis: Generally, as you increase the temperature the time taken for the clinistix to change colour decreases, graph 1 shows this. At 20ºC it has taken 275 seconds for the clinistix to change colour, but once this temperature is doubled to 40ºC it has taken only 120 seconds for the clinistix to change colour. As you increase the temperature the rate of reaction increases. On graph 2 you can see the general trend, the graph line is going upwards. At 20C the rate of reaction is 0.36, at 40ºC it has risen 0.47 to 0.83, at 60ºC it has risen 1.31 to 1.67 from 20ºC. Conclusion: As you increase the temperature the rate at which the lactase enzymes hydrolysed the lactose into glucose and galactose increased, and did not seem to slow down at 60ºC. When lactase is immobilised it becomes more stable and so can work more efficiently at higher temperatures without denaturing, the material the enzyme is attached to gives the enzyme strength. This means that enzymes can keep their shape (i.e. do not denature) past their normal tolerant temperature. More energy is required to break the hydrogen bonds holding the secondary and tertiary structure of the enzyme, when these are broken the active site loses its shape and the substrate can no longer bind to it and the reaction is no longer catalysed. Normally lower amounts of energy are required to break these bonds so the enzyme will become denatured a lot quicker than when it is immobilised. The extra energy required when the enzyme is immobilised is to break the enzyme?s bonds and the immobilising agent together. Evaluation: My results were neither very accurate nor reliable. My accuracy was not good, because I used a quick method to test for any glucose sugars (using clinistix), instead of carrying out a Benedict?s test and using a colorimeter, this would take longer but would be a lot more accurate. The method I used is inaccurate as it is difficult to see the colour change (due to the milk) and time when this colour change exactly occurs (colour change takes time, it is not instant). My results were unreliable, because I only took one set of results, not two or three. If I had taken more sets of results, my results would have been reliable. One problem I encountered while doing this experiment was that the temperature was fluctuating as I had used a beaker of water upon a Bunsen burner not a water bath. This interfered with my results. To solve this problem I can next time use a water bath. The biological significance of this experiment is important especially in industry due to the factors mentioned in my background information. To extend my investigation I could use a control (unimmobilised lactase enzymes) and I could compare immobilisation on other enzymes.

The Effect of Temperature on Lactase Immobilised Enzymes in an Enzyme Controlled Reaction 8.9 of 10 on the basis of 2702 Review.