Effect of Temperature on Immobilisation of Pectinase
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The use of a catalyst is to lower the required energy level for a reaction to take place, the activation energy. This means less energy can be put into a reaction, saving money, making it an economically viable solution for a business with its primary aim being making money. Catalysts are also reusable if used in the correct state (one that can be separated from our product once the reaction has reached its end, in our case the solid Alginate beads in a liquid product, which can be simply filtered by passing the juice/enzyme mixture through a filter), so can be effective over and over, becoming more cost effective the more times they are used, making them a wise investment to increase the yield of the product, in this case, the quantity of fruit juice from a fruit pulp.
Pectinase plays a vital role in the extraction of juice from fruit. Pectinase breaks down pectin, the glue like substance that sticks plant cells to each other so it can extract the juice from the fruit. Pectinase being an enzyme, a biological catalyst, naturally has a range in which it is most effective determined by its 3D globular protein structure. The 3D globular protein structure is made up of 3 different levels of structure. The primary structure of amino acids (a chain of amino acids bonded to each other by peptide links), is folded into an alpha helix or beta pleated sheet, which are then interwoven between each other, and joined by Hydrogen bonds, Disulphide bridges, and other types of bond depending on the make up of the R group. The constituents of the R group determines the type and strength of the bonds and so the denaturation point of the enzyme, the point at which these bonds are broken or disrupted, and the active site to which our substrate (Pectin) can no longer fit, so the enzyme cannot work to break it down again. So our pectinase enzyme will become ineffective. If we can overcome the weakness of these polypeptide chain interactions by protecting the enzyme so its bonds are less easily broken, and use a temperature stable enzyme, the prospects for a business can be very appealing. Firstly, if we can increase the temperature of our reaction, the collision theory states that this will give our particles more energy, so more collisions, so a faster reaction. Giving our particles more energy will cause them to move around at a faster rate, moving around at a faster rate they are more likely to collide with other reactants at the required energy for the reaction to happen. If we raise the overall temperature, we raise the overall energy these particles have, so more collisions between particles will happen. When particles collide they react, if we increase the number of particles colliding, we can increase the rate.
If we can harness the use of both increased temperature and a catalyst, the rate and yield would be vastly increased, and a greater yield means more profit for a company. It also would lead to a sterile environment, so an uncontaminated product.
One way we can both use a catalyst and increase temperature is by immobilising the enzymes. This suspending them in an Alginate bead, which is temperature resistant to up to 300ï‚°C . Surrounding our enzyme, a 3D globular protein held together by hydrogen bonds, ionic bonds and disulphide bridges, in Alginate will secure and support the 3D structure hence making sure the active site is unaltered when temperature is raised (temperature that would normally have enough energy to break these ionic, disulphide and hydrogen bonds). This means the active site where our substrate sits, and the enzyme will function as it would in a free enzyme before denaturation. It also means the catalyst is no longer a liquid, it is in a different state to the substrates, and so a simple filtration can separate the two for easy reuse. This saves money as no more has to be spent on catalysts. It will however lead to a decrease in surface area, as some enzyme is entrapped in the centre of these beads, which could cause a slower rate than a free enzyme, but as temperature is increased, these beads will work consistently as well, not being affected by the denaturation point of the enzyme. In fact they should continue to work faster as temperature increases above the normal denaturation point, as successful collisions between substrate and active site will continue to increase.
Null Hypothesis - There is no significant difference between using a free and an immobilised enzyme at 70ï‚°C in the production of juice from fruit.
Experimental Hypothesis - There is a significant difference between using a regular and immobilised enzyme at 70ï‚°C in the production of juice from fruit
(The temperature was selected from the graph in the first half of my experiment)
The independent variable is whether the enzyme is free or immobilised, and the dependant variable is the volume of product produced. I controlled variables such as the temperature at which the experiment occurred (after selecting this temperature to be the most suitable based on my trial experiments), the concentration of pectinase used in each experiment, more enzyme would lead to a faster rate, the size of the beads was an important factor to ensure validity in my experiment, a factor I discuss in more detail below. I also had to use the same type of fruit, I settled on Williams pears, different varieties of the same fruit may give more or less product when exposed to the enzyme, so I had to be consistent with this. I also used the same quantity of fruit each time, accurately measured by using a mass transfer table, to ensure that the same mass of fruit was used each time, as obviously more fruit would give more product, whatever the conditions. Controlling all these variables and only changing my independent variable increased the validity of my experiment.
Equipment list and Justification
Williams Pears. Differences in varieties of pear could react differently to Pectinase. Just as important is having fresh ripe fruit, for the same reason. I also used Granny Smith Apples in my initial tests.
Pectinase enzyme. Upwards of 30ml depending number of repeats/temperatures chosen. stored appropriately (refrigerated between 2-5ï‚°C) and within date to ensure the enzyme has not previously denatured so would give us false results were we to use it in our experiment. Can test the activity your own enzyme against a recorded data value to check it is still fully functional.
Several sheets of Muslin, at least 3 to allow you to continue with the experiment without waiting for the Muslin to be clean/dry
Filter funnels and measuring cylinders (50ml) all used to collect and measure the yield of product
Calcium chloride solution. 100ml, can be reused
Knife, Peeler, Chopping board and Food processer/Blender to prepare my fruit to give us the greatest yield (absence of skin which is unaffected by Pectinase)
Tea strainer to separate immobilised Pectinase from product
Syringe, nibs (tips of the syringe from which the liquid is expelled, the measurement is the diameter of the nib) 0.8mm (trial) 0.4mm (final). 10ml, as we are using 10ml of enzyme/Alginate solution ( 1:4 ratio) so any larger syringe would give us less accurate data as a larger syringe would have a greater percentage error, due to being suited to measure a larger amount of volume. A smaller nib can form the same size bead more consistently as it takes less Alginate for the bead to fall under it's own weight, so the margin for error is smaller.
Electronic balance for measure of our fruit pulp, we can make the measurement of fruit more accurate by firstly measuring the vessel and fruit together, then just the vessel by itself to insure 50g of our fruit was transferred, as obviously more or less fruit would give more or less juice respectively, not a variable we wished to change
Stopwatch for keeping time of exposure to enzyme consistent, we wish for the enzyme to act on each for the same amount of time on each sample.
Water baths 20°C to 60°C (approx), increments of 2-3 ï‚°C
Preparation of Immobilised Enzymes
I mixed 8ml of Alginate and 2ml of Pectinase in a beaker, which could also be larger amounts in the same 1:4 ratio. I extracted 10ml of this into a syringe. I prepared a beaker of 100ml calcium chloride solution, by dissolving calcium chloride in distilled water, then using a magnetic stirrer to mix the solution. It was suggested I use 0.2 molar concentrations here, as a change in pH can give more or less stable beads.  Using a 0.8mm nib on the syringe, I slowly formed a bead on the end of the nib over the calcium chloride until it falls into the solution under its own weight so that all beads will be the same size. I continued until all 10ml of solution has been passed into calcium chloride solution and I had 10ml equivalent of immobilised enzyme. I extracted the beads I made from the beaker using a tea strainer. Separating calcium chloride and immobilised enzyme.
These beads contain a reusable catalyst so can be used several times, to reuse after an experiment they must be filtered out, which saving making more, reducing time and cost. However, it should be noted that several sets of beads should be used, as it has been reported the repeated use of beads leads to a decrease in enzyme activity as noted in a separate experiment  . Due to the porous nature of the Alginate beads, some leakage does occur of the enzyme, and obviously fewer enzymes will give less of an effect. It is also suggested that at each session the experiment takes place, a new set of Alginate beads should be used, as storage over time also leads to reduced enzyme activity.  We must also be careful with the concentration of enzyme used, as too high a concentration may damage our Alginate beads causing them to be ineffective, and offer little protection to the immobilised set of enzymes.
Accuracy of Immobilisation of Pectinase
When producing the beads in the previously stated method, accuracy in the size/shape of these was vital to ensure validity to my whole experiment. In order to maintain accuracy throughout my experiment, I needed to ensure the sides of the Alginate beads I produced were equal. If I did not, and too many beads were disproportionally small or large, ratio of the surface area of the bead to the contents of the bead would be too great; fewer enzymes would be exposed to the substrate, causing it to be ineffective, excluding a significant part of the enzyme volume from the experiment, in a given bead. If this was not controlled, it could seriously affect my results, as an inaccurately produced group of beads could cause the enzyme to work inefficiently in a given reaction, causing my results to not give a true reflection of the actual effect of the immobilised enzyme. I therefore needed to test, to make sure they were of equal sizes, and took a random sample of the beads I produced (I reused my beads each time, so I only needed to take a sample from 1 set of beads, in order to not cause a bias between different groups of beads) to ensure the beads maintained an average size, none particularly large or small. I had someone else who was not aware of my aim to select the beads at random from the set to make sure my calculation of the bead sizes was not affected by my experiment bias, as I may, even when making a random choice, subconsciously pick beads I see the same size so my creating of the beads seems more accurate, and experiment more accurate. I estimated that a 10ml mixture (8ml of sodium Alginate, 2ml of Pectinase enzyme) using a 0.4mm nib produced approximately 80 beads. Hence I took a sample of 8, a 10% sample of the population of beads to be measured. The beads were measured using a micrometer, measuring the diameter of the beads, which I took to be the same all the way round the bead, as the beads were spherical when produced.
Using the micrometer gave me the measurements 2.5mm, 2.7mm, 3.0mm, 2.8mm, 3.5mm, 4.8mm, 2.9mm and 2.8mm. An average of 2.88mm excluding 4.8mm, as this value was clearly an anomaly in my data. If I calculate the average volume of my bead to be 4/3 x pi x 1.44³ = 12.5mm³ (this assuming the beads are perfectly spherical, using the formulae 4/3 x pi x r³), then divide my volume used by the average size I should get my total number of beads, 1000/12.5=79.95. Taking a 10% sample from 80 is sufficient to have a consistent average proving my bead size has remained consistent.
I chopped 1 apple into small pieces, 5mm x 5mm x 5mm approx. I weighed 50g of apple into 1 beaker. In one beaker I placed free enzyme (2ml of Pectinase, 8ml of distilled water), in the other place 10ml of Immobilised enzymes by the preparation earlier stated. I placed into water baths of given temperatures, from 20 ï‚°C upwards letting them incubate for 20 minutes. I selected 20 minutes after preliminary experiments showed the reaction has gone fully at this point. I had the same volume of juice after 20 minutes, to an otherwise identical solution that had been incubated for 30 minutes. Therefore after 20 minutes the reaction has finished. I noted the temperature used. After I filtered the solutions using coffee filter paper, I noted the amount of juice collected in both immobilised and regular enzyme solution. (Filter enzymes at this point to reuse). I then repeated the above test, increasing temperatures by between 2-3 degrees, on each experiment, noting the product collected. I then selected a final temperature to use in the statistical test to find a significant difference.
This data should allow me to calculate the effectiveness of the immobilised enzyme, and the normal denaturation point and how the immobilised enzyme has been unaffected by this due to the enzymes 3D globular structure being supported in the bead. The statistical test I selected to determine whether there is a correlation in my data was the Mann-Whitney U test; this relied on me having between 6-20 sets of data, between two different variables, in my case - the immobilised and regular action of pectinase. I first found the ideal temperature to test at to find a significant difference, then would gather at least 8 sets of data from this temperature.
The biggest change was replacing apples with pears, which were puréed instead, as well as being peeled. I found pears gave a greater yield than apples in otherwise identical experimental conditions. I chose for the greater yield, as it is easier to see a difference graphically when the volumes produced are around 20ml, rather than 5ml. The equipment is only accurate to 0.5ml, 10% difference around 5ml may not be observed, whereas if this change occurs when our volumes are at 20ml, the equipment will allow us to see this chance. Peeling and blending increased the surface area of the fruit, so a bigger contact with the enzymes; greater interaction between the surfaces leads to a faster yield. The Pectinase is ineffective with the waxy outer layer of fruits, so I excluded peel from my experiment to gain better results. Using the smaller nib to produce my immobilised enzymes gave me a greater surface area to volume ratio, so more of the fruit is exposed to the enzyme, making it as similar as possible as the liquid. Although more time consuming to create the beads, as they were smaller and more delicate to make with this nib, the increased surface area should give better results as it has increased contact with the fruit. The smaller beads giving me more consistent results, as it is much easier to produce a set of smaller beads the same size than a set of large beads the same size, there is a greater chance of error with the large beads. The smaller beads also had a greater volume:SA ratio, so increased my yield and made it easier to identify a correlation/apply my results to the stats test. Muslin replaced the coffee filter paper. Muslin is a fabric, so does not soak up and clog like the filter paper did, it has a finer more effective mesh. It is also reusable, unlike the filter paper. I also lowered the upper temperature I was going to use, as it was hard to obtain safely with our equipment and unnecessary to the experiment. All these will be implemented to improve my experiment by reducing time/materials used, safety and to improve accuracy. I also took the opportunity to alter my experiment for the better, by using a mass table to calculate mass of fruit actually transferred. I would measure my initial mass (fruit + vessel) then just the vessel, and subtract the values, making sure that this value I calculated was 50g, so 50g was actually being used in my experiment.
I peeled then chopped several Williams Pears into small pieces, 5mm x 5mm x 5mm approx, quickly, to ensure freshness and no browning of the fruit
I weighed 50g of Pear into a beaker using the mass table method as previously stated, and then pureed this.
In one beaker I placed regular enzyme (2ml of Pectinase, 8ml of distilled water), in the other place 10ml of Immobilised enzymes by the preparation earlier stated.
I placed into water baths of given temperatures, from 20 degrees upwards letting them incubate for 20 minutes. Note the temperature used.
Filter the solutions using Muslin, note the amount of juice collected in both immobilised and regular enzyme solution. (Filter immobilised enzyme at this point, and remember to subtract 10ml from our regular enzymes product, as the liquid enzyme also passes through the Muslin.)
I repeat for at least 3 times for each temperature to assess reliability, and took an average
Repeat steps 1-6, increasing temperatures by between 2-3 degrees, on each experiment, noting the amount product collected
I continued up to 70 degrees. At this temperature I repeated 10 times for both immobilised and free enzymes.
I repeated 10 times as between 6-20 sets of data are required to apply to the Mann Whitney U test, at p=0.05. Using 10 sets of data is enough to see statistically if there is a significant difference and so if I can disprove my null hypothesis.
Wear gloves and goggles if
needed when handling calcium chloride
Having a maximum risk level of 8/25, I and my teachers decided my experiment was safe to do.
Amount of Juice produced (ml) when immobilised and free Pectinase are
subject to rising temperatures
Temperature of water bath in which reaction occurred
Volume of Product when using Immobilised Enzyme
Volume of Product when using Free Enzyme
Greatest difference occurs at 70°c
When this data is placed in the form of a line graph it is clear to see that the regular enzyme having gives a greater yield before it's denaturation point, and the clear difference in product at 70°C, the temperature at which I will be testing the significance of this difference.
Series 1 - Immobilised Pectinase
Series 2 - Regular Pectinase
With the clearest difference visually being at 70°c, I decided I would take this value to be the one I tested to find if there was a statistical difference.
The experiment was repeated, as before, but only at 70°c. I took 10 repeats so I had sufficient data to test in the Mann-Whitney U statistical test at p=0.05. 5% probability level sufficient enough for me to be sure my results were not down to chance.
Volume of Juice (ml) produced from 50g of Williams Pears at 70°C when treated with pectinase in immobilised and free forms.
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