To investigate and compare the activity of immobilised enzymes against enzymes free in solution at a range of different concentrations.
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Biology Coursework Planning Aim: To investigate and compare the activity of immobilised enzymes against enzymes free in solution at a range of different concentrations. Introduction: Lactose: C12H22O11, found in the milk of mammals, is a disaccharide composed of one glucose and one galactose molecule joined by a beta 1:4 linkage. C12H22O11 C6H12O6 + C6H12O6 Lactose glucose galactose Draw out structure from sheet . To utilise lactose an organism must posses a specific enzyme (lactase) which catalyses the hydrolysis of lactose into glucose and galactose. To fully utilise lactose a organism must also have a second enzyme which subsequently converts galactose into glucose. Among adult humans, the ability to digest lactose is actually relatively rare (confined particularly to whites of Northern European ancestry) and lactose intolerance occurs due to an absence of production of the human lactase digestive enzyme which catalyses the hydrolysis of lactose into its constituent monomers. Enzymes Enzymes are biological catalysts. A catalyst is a substance, which speeds up a reaction without getting used up itself. Industrial processes use catalysts to speed up production, reducing the need for high temperatures and pressures. The human body temperature is 37C, and without catalysts the reactions would be too slow to sustain life. Since there are thousands of different reactions happening in cells, and each requires a specific enzyme, enzymes must be made of a type of chemical which can vary enormously in structure, amino acids. There are roughly 20 different amino acids, which can be linked in infinite amount of ways. This means that the primary and tertiary structure of enzymes can show the variability needed to achieve specificity, particularly upon the shape of a small part of the enzyme molecule where the enzymes actually come in contact with the substrate molecule, the active site. This is a small 'cleft' on the enzyme surface, where certain amino acid residues are exposed. The substrate molecule fits into the active site and interacts with these amino acids by ionic and hydrogen bonding, forming and enzyme-substrate ...read more.
Perhaps there was a temperature increase, as the milk had been left out during the experiment when the 10% result was taken, making the rate faster, so that the 100% and 10% result had little difference between them. This could be improves in the final experiment by measuring the temperature and pH of the milk before the experiment begins, in both the immobilized and free in solution lactase enzymes. Another reason, that could have caused, this result, was that the syringe barrel was not cleaned out properly between results, which might have meant that different enzyme concentrations, may have been mixed. Other areas, that I think would improve the experiment, is when making the beads leave them for a set amount of time to harden so that they do not clump together, and I should make sure that the mixtures of sodium alginate and lactase are mixed together well. The beads often came out at different sizes, especially when forming my first set of 100% concentrated beads, which might have meant that small beads were penetrated faster, which meant more enzyme substrate complex's were formed quicker, increasing the rate. The number of beads, therefore also varied in the syringe barrel, within the 5cm3 mark. I think I need to make sure the beads out into the barrel, are all of similar size, this will also prevent beads getting stuck in syringe outlet restricting the flow of milk droplets. I think that more than one set of results should be taken for each reading so that an average can be taken, for a fairer experiment, decreasing the chance of human error, and making sure that results, can be determined as anomalous fairly, such as the 0.1% results found above. Final experiment: Apparatus: * 4cm3 Lactase (2cm3 for the immobilised enzymes and 2cm3 for the enzymes free in solution) * 8cm3 Sodium Alginate solution, made up in distilled water * 100cm3 of 2% calcium chloride solution * Clinistix * 10cm3 plastic syringe and syringe barrel (with measurement markings) ...read more.
This could be improved, by using more advanced type of glucose detecting sticks to measure the percentage of glucose conversion. This will give a quantitative, which would produce more accurate tables and graphs. I could use a set time for to check how much is converted. This would prevent problems such as interference of the beads and lactose and galactose molecules, blocking the glucose molecules from the end of the syringe outlet. * Another difficulty, was in the rate of drops flowing from the syringe barrel, was hard to control, alternative apparatus, should be looked into, e.g. a burette * I could look further into the structure and weights of the molecules produced and used, to see if e.g. weight of the molecules, effects how fast they reach the end of the syringe, perhaps, if glucose is lighter molecule than galactose, than it will not sink to the bottom of the syringe as quickly. This might mean that I should measure glucose conversion, by sticking the glucose stick into the beaker beneath the syringe outlet, after it has been stirred at a certain time, rather than the drops from the syringe outlet. * I could do more detailed percentages especially exploring between 10% and 100% where the largest gradients were produced. The experiment could be explored further, by changing a different variable, such as temperature or pH, to determine and prove these effects experimentally, to broaden my understanding of lactase enzymes further. I could explore what the activation energy, denaturing and optimum rate temperature/pH would be. Health and safety had to be considered in this experiment, here are some of the guidelines I followed, as calcium chloride is irritating to the eyes, skin and respiratory system, we tried to make minimum skin contact and limit the possibility of inhalation, by careful and conscious handling of the products. Small amounts of lactase were left out for use, so there was none left over, so to avoid any drying up and causing dust formation, which could provoke asthma or hay fever, any spillages were cleared up quickly with water. ...read more.
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