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Investigate the effect of temperature on the activity of an immobilised enzyme.

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Introduction

Investigate the effect of temperature on the activity of an immobilised enzyme. Introduction B-galactosidase (lactase) catalyses the hydrolysis of Lactose to glucose and galactose. Milk, or whey derived from it, may be treated using either free of immobilised enzyme in a batch reactor. Alternatively, the substrate may be passed continuously through a column containing immobilised enzyme. The product of this treatment is lactose reduced milk. Although beta-galactosidase is widespread among bacteria, it is not universal, and is rarely found in eukaryotes, where different enzymes (with different specific activities) accomplish the digestion of lactose. In humans, however, many adults are deficient in lactase, the intestinal enzyme that accomplishes digestion of the same substrate - as many as 70-90% of adults in most parts of the world are lactose-intolerant for this reason. Hypothesis My Hypothesis is that the immobilised enzyme will be most stable at the optimum temperature for enzyme activity, about 40 oC. The enzyme should show a decrease in stability at more extreme temperatures, 20 oC and 60 oC. Immobilising the enzyme means it should stay fairly stable at slight changes in temperature, 30 oC and 50 oC, because of the protection offered by the inert matrix. Enzymes are globular protein molecules that are responsible for all the biochemical reactions within cells. These reactions are essential for the life of the organism. Enzymes act as catalysts, substances that accelerate the rate of a chemical reaction, by reducing the activation energy necessary to initiate the reaction. During an enzyme-mediated reaction, the substrate physically combines with the enzyme at its active site, allowing the substrate to be converted to a new product or products. The enzyme, however, is not changed by the reaction (and thus is technically not a reactant in this reaction) and is recovered to participate in other reactions. Because the same enzyme molecule can be used over and over, only small quantities of enzymes are needed in the cell. ...read more.

Middle

Calcium chloride: Low risk avoid contact with eyes. Milk: Very low risk. USE EYE PROTECTION Plan: Pilot experiment, Range of readings In order to determine suitable temperatures, for the immobilised lactase beads to be heated to, I carried out rough preliminary experiments heating the beads to different temperatures, I used the range I intended to use, 20oC, 30oC, 40oC, 50oC and 60oC respectively, and other sets of temperature ranges, 25oC, 30oC, 35oC, 40oC and 45oC, and 35oC, 40oC, 45oC, 50oC and 55oC. I then carried out experiments to determine which set of temperatures would give the best results. Results of pilot tests, Range of readings Using range of temps 20oC, 30oC, 40oC, 50oC and 60oC Temperature (oC) Glucose concentration (mmoldm-3) 20oC 1/10% (5.5) 30oC 1/4% (14) 40oC 1/4% (14) 50oC 1/4% (14) 60oC No Result Using range of temps 25oC, 30oC, 35oC, 40oC and 45oC, and 35oC Temperature (oC) Glucose concentration (mmoldm-3) 25oC 1/4% (14) 30oC 1/4% (14) 35oC 1/4% (14) 40oC 1/4% (14) 45oC 1/4% (14) Using range of temps 35oC, 40oC, 45oC, 50oC and 55oC Temperature (oC) Glucose concentration (mmoldm-3) 35oC 1/4% (14) 40oC 1/4% (14) 45oC 1/4% (14) 50oC 1/4% (14) 55oC 1/10% (5.5) From the results I obtained it was clear that the first range of temps, 20oC, 30oC, 40oC, 50oC and 60oC respectively gave the clearest and most varied results, and I could draw more conclusions from them, I therefore have chosen to use these range of temperature in my practical. Method Firstly, I immobilised 2cm3 of lactase (Beta galactosidase) by mixing it in a beaker with 8 cm2 of 2% sodium alginate solution, then adding this mixture drop wise to a new beaker of 100 cm3 of 1.5% Calcium chloride solution. I allowed the immobilised enzyme pellets to harden for a few minutes, then rinsed them thoroughly with distilled water before use. I made 5 batches of these immobilised enzyme beads. ...read more.

Conclusion

The other major limitation was the temperature regulation, the water baths were unreliable and took a lot of time to heat the immobilised enzyme beads to the required temperature, and even then the temperature couldn't be kept exact, there were especially problems heating the water to 60oC and cooling it to 20oC. If more advanced water baths that can heat and cool water to a desired temperature quickly were available this could have improved the accuracy of my results however there were not available, therefore I solved this problem by adding boiled water to the water in the beaker to reach the required temperature, however it was still very time consuming and still slightly inaccurate. Time constraints limited the experiment greatly as there was no time for further work or to investigate anomalies, this resulted in drawing conclusions from guess work. However, if I had carried out the further experiments as I had wished I would have far exceeded the maximum time allocated to the experiment. Overall the investigation did provide significant evidence to support my hypothesis that, the immobilised enzyme will be most stable at the optimum temperature for enzyme activity, about 40 oC. The enzyme should show a decrease in stability at more extreme temperatures, 20 oC and 60 oC. Immobilising the enzyme means it should stay fairly stable at slight changes in temperature, 30 oC and 50 oC, because of the protection offered by the inert matrix. Evaluation: Further work If the time were available, I would have liked to have carried out a similar experiment with free lactase to make comparisons on the immobilisation procedure on temperature stability, I would have also wished to have investigated why the enzyme denatured so quickly at high temperature - even though it had been immobilised - to see if this was the nature of the enzyme or an experimental flaw. Also a greater range of temperatures would have given improved results to draw conclusions from, investigating the effects of colder temperatures on the enzyme activity. James Casson ...read more.

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