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Immobilised enzymes biology practical yeast andglucose
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Thermophilic fungi are known to produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we report the characterization of a protease produced in state-solid (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus My-celiophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a media composed by a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. The crude enzyme obtained by both SSF and SmF displayed equivalent optimum temperature at 50ºC, however the optimum pH shifted from 7 (SmF) to 9 (SSF). The alkaline protease produced through solid-state fermentation (SSF), was immobilized on beads of calcium alginate, allowing to carry out comparative analyses of free and immobilized proteases. It was observed that both optimum temperature and thermal stability of the immobilized enzyme were higher then for the free enzyme. Moreover, the immobilized enzyme showed good stability for up to 7 reuses. Keywords: alkaline protease, immobilized enzyme, Myceliophthora sp., solid state fermentation, thermophilic fungus. Enzymes have a wide variety of biotechnological, biomedical, and pharmaceutical applications. Proteases constitute are the most important group of commercially available enzymes (Joo et al., 2002); they account for about 65% of the worldwide sale of industrial enzymes in the world market (Johnvesly and Naik, 2001). Microorganisms are an important source of proteases mainly due to t... ... middle of paper ... ...erature and pH on free and immobilized enzyme stability The thermal stability was investigated by measuring the enzyme activity after keeping the enzyme solution for 1 h in the absence of substrate, at temperatures between 30 and 70°C in the absence of the substrate. The effect of pH on the stability was analyzed by incubating ali-quots of the enzyme at room temperature at pHs from 8 to 11 at 1, 3, 5 and 24 h. Remaining protease activity was determined at optimum pH and temperature. Reusability of protease immobilized in the alginate beads The initial activity of the immobilized enzyme was measured and the conjugate was then sub-jected to cycles of repeated use. The results of pH, temperature and reusability stability of free and immobilized protease are presented in a normalized form, with the highest value of each set being assigned the value of 100 % activity.
Living organisms undergo chemical reactions with the help of unique proteins known as enzymes. Enzymes significantly assist in these processes by accelerating the rate of reaction in order to maintain life in the organism. Without enzymes, an organism would not be able to survive as long, because its chemical reactions would be too slow to prolong life. The properties and functions of enzymes during chemical reactions can help analyze the activity of the specific enzyme catalase, which can be found in bovine liver and yeast. Our hypothesis regarding enzyme activity is that the aspects of biology and environmental factors contribute to the different enzyme activities between bovine liver and yeast.
Finally, the last part of the experiment examined the enzyme activity at different pH levels. Four sets of 11 tubes were set up in this part. The procedure for this part is the same as before, but 4 other buffers were substituted for the standard pH 7.3 phosphate buffer. Set A used the 5.5 pH buffer while set B used the 6.5 pH buffer. The buffer of pH 8.5 was used for set B and for set D the pH was 9. The absorbance readings for 4 sets were taken and recorded in table 13. Using the linear equation that the best-fit line gave for each set, the Km and the Vmax of each set were determined. Then, table 15 was made by dividing the Vmax by the Km. of the four pHs. The Vmax and Km of the control set were also used to make
The shape of the molecules is changing and so the enzyme molecules can no longer fit into the gaps in the substrate that they need to and therefore the enzymes have de – natured and can no longer function as they are supposed to and cannot do their job correctly. Changing the temperature: Five different temperatures could be investigated. Water baths were used to maintain a constant temperature. Water baths were set up at 40 degrees, 60 degrees and 80 degrees (Celsius). Room temperature investigations were also carried out (20 degrees).
The purpose of this experiment was to discover the specificity of the enzyme lactase to a spec...
The Effect of Temperature on the Activity of the Enzyme Catalase Introduction: The catalase is added to hydrogen peroxide (H²0²), a vigorous reaction occurs and oxygen gas is evolved. This experiment investigates the effect of temperature on the rate at which the enzyme works by measuring the amount of oxygen evolved over a period of time. The experiment was carried out varying the temperature and recording the results. It was then repeated but we removed the catalase (potato) and added Lead Nitrate in its place, we again tested this experiment at two different temperatures and recorded the results. Once all the experiments were calculated, comparisons against two other groups were recorded.
In this experiment as a whole, there were three individual experiments conducted, each with an individualized hypothesis. For the effect of temperature on enzyme activity, catalase activity will be decreased when catalase is exposed to temperatures greater than or less approximately 23 degrees Celsius. For the effect of enzyme concentration on enzyme activity, a concentration of greater or less than approximately 50% enzymes, the less active catalase will be. Lastly, the more the pH buffer deviates from a basic pH of 7, the less active catalase will be.
Alkaline Phosphatase (APase) is an important enzyme in pre-diagnostic treatments making it an intensely studied enzyme. In order to fully understand the biochemical properties of enzymes, a kinetic explanation is essential. The kinetic assessment allows for a mechanism on how the enzyme functions. The experiment performed outlines the kinetic assessment for the purification of APase, which was purified in latter experiments through the lysis of E.coli’s bacterial cell wall. This kinetic experiment exploits the catalytic process of APase; APase catalyzes a hydrolysis reaction to produce an inorganic phosphate and alcohol via an intermediate complex.1 Using the Michaelis-Menton model for kinetic characteristics, the kinetic values of APase were found by evaluating the enzymatic rate using a paranitrophenyl phosphate (PNPP) substrate. This model uses an equation to describe enzymatic rates, by relating the
Many factors, for example, pH and temperature affects the way enzymes work by either increasing the rate or determining the type of product produced (). The report, therefore, analyses the effects of the enzyme peroxidase in metabolic reactions and determining its optimum temperature in the reactions.
This indicated that the effect of high temperature on the activity of peroxidase was irreversible and so if the optimum temperature was restored the enzyme activity will not increase again because denaturation resulted in a permanent change in the shape of the active site of the peroxidase enzyme. In conclusion, the results of this experiment supported the hypothesis that enzymes including peroxidase enzyme are sensitive to temperature changes[George
In this lab, it was determined how the rate of an enzyme-catalyzed reaction is affected by physical factors such as enzyme concentration, temperature, and substrate concentration affect. The question of what factors influence enzyme activity can be answered by the results of peroxidase activity and its relation to temperature and whether or not hydroxylamine causes a reaction change with enzyme activity. An enzyme is a protein produced by a living organism that serves as a biological catalyst. A catalyst is a substance that speeds up the rate of a chemical reaction and does so by lowering the activation energy of a reaction. With that energy reactants are brought together so that products can be formed.
My prediction was very accurate as there were little products at room temperature, and according to my results the optimum temperature was only about 1ºC higher than my prediction. I also correctly predicted that the enzymes would denature after 40oC and that the graph would be exponential. Conclusion The graph starts with little carbon dioxide production at the low temperatures.
From looking at the results I can conclude that when the pH was 3 and 5. No oxygen was produced, therefore no reactions were taking place. This was because the pH had a high hydrogen ion content, which caused the breaking of the ionic bonds that hold the tertiary structure of the enzyme in place of the syringe. The enzyme lost its functional shape.
The pectinase enzyme will work most effectively at a constant room temperature 25-35 degrees Celsius.
Purpose: The purpose of this lab is to explore the different factors which effect enzyme activity and the rates of reaction, such as particle size and temperature.
Introduction: Purifying proteins is an important part of biology because it can help identify the function of that protein. Once a protein’s function has been identified, it can be manipulated to see how the function would change if the protein was changed. A common way to purify a protein is through Ion Exchange Chromatography, which is where charged proteins will bind to the beads in the column to purify it from the solution (Berg JM, 2002). The purpose of this experiment is to use Ion Exchange Chromatography to purify cellulase.