Bio1022 – Practical 3 Aim: An investigation of amylase activity on the different stages of barley seeds development Introduction Metabolism involves a series of chemical reactions which allows the organism to maintain its structure, increase its biomass, reproduce and react to their surroundings. In a dormant barley seed, starch is stored in the endosperm as a source of energy storage (King, Reiss & Roberts, 2001). Starch is subsequently broken down into its constituents, being glucose. Hence, the role of amylase within this reaction is to hydrolyze starch to maltose (Reaction 1). Lastly in order to further breakdown maltose into glucose requires another enzyme, glucosidase (Reaction 2). Reaction 1: Starch …show more content…
After that the seeds were crushed to a fine paste with the 10ml of buffer added as the seeds were crushed, the paste is then subsequently filtered into a measuring cylinder. The solution collected after filtration is the amount of amylase extracted solution (AE), with its volume being recorded. The amylase extracted solution(5ml) is then transferred into a beaker, then a five-fold dilution of the amylase extract using 20ml of buffer. Resulting in 25ml of diluted amylase extract (DAE). A control extract is prepared (5ml of DAE) to a test tube, which is then placed in boiling waterbath for 10minutes, after 10minutes remove the control extract and leave it to cool at room temperature. In order to determine the amylase activity, one drop of iodine is dropped into 21 labelled wells on the ceramic test plates. A reaction mixture is prepared, 5ml of buffer and 1ml of 0.5% starch solution to a test tube. Extract one drop from the reaction mixture to the well labelled T. Turning blue-black indicating the presence of starch in the reaction mixture. Add 1 ml of diluted amylase extract to the reaction mixture. Starting with well number 0, add one drop of amylase reaction mixture to a different well each minute until the achromic point is reached (no color change). Repeat the experiment using boiled control extract instead of DAE for the determination of amylase …show more content…
As stated in the thesis “Amylase activity in dormant and germinating seeds” the amylase activity of whole seeds rises very sharply to a maximum and remains constant during the germination period. However, this discrepancy can be attributed to the age of the seeds as older seeds have significantly lower amylase concentration after long period of dormancy (Ernst, 1971). Another reason may be due to experimental errors during extraction of the amylase from the barley seeds, for example the seeds that are not grinding finely can lead to a decrease in amylase concentration. Lastly only germinating and whole barely seeds showed the presence of maltose, indicating only within these two there is amylase present which actively hydrolyzing starch into maltose, as per reaction 1 stated above. As dormant seeds have amylase concentrations that are far too low to be detected by this type of assay, as during dormancy energy demands for this state is considered to be zero. Hence the amylase concentration in the dormant seeds are far too low to be detected in the Benedict’s reagent test (Ernst,
Fermentation test is used to determine if unknown #398 uses any oxygen to ferment carbohydrates and acids. Oxidation tests were used to determine if unknown #398 metabolizes carbohydrates and acids by cellular respiration. Both tests are observed by inoculation of unknown #398 into 3 sugar broths: lactose, glucose, and mannitol and 1 citrate (Citric acid) slant. Fifth test, Hydrolytic and Degradative reactions is used to determine if unknown #398 contains enzyme, amylase that hydrolyzes starch after streaking on a starch plate. Next test, inoculation of a urea broth and is used to determine if unknown #398 contains urease that hydrolyzes urea.
Abstract This experiment showed that temperature, concentration and pH all affect the rate of enzyme reaction differently. Enzymes are very important in organisms and therefore understanding how and why they work the way they do in specific conditions is crucial. The results showed that an increase in temperature would also increase the reaction rate, until a temperature that was too high, where the enzymes began to denature and therefore the rate of reaction was slowed down. As concentration was increased, the reaction rate continued to increase.
Starch solution is then placed into the test tube at a quantity of 5 mL. 5 drops of Lugol’s Iodine solution is added to the test tube. If the color changes, then it is known that starches are present in the solution. Proteins are next tested. In order to do this, 5 mL of gelatin solution is added to the test tube. 10 drops of Biuret’s reagent are added to test for protein.
Sucrase activity increases with increasing sucrose concentration Materials and Methods Effect of pH on Enzyme Activity 1. Dependent Variable amount of product (glucose and fructose) produced 2. Independent Variable pH 3. Controlled Variables temperature, amount of substrate (sucrose) present, sucrase + sucrose incubation time Effect of Temperature on Enzyme Activity 1.
Introduction: Experiments of the past have confirmed enzyme activity is a significant determinate in the activity of the α-casein with β-casein (Xiaoyu, Yongkang, and Zheng 2012). It’s already been shown before that there are specific enzymes such as Psychromonas ingrahamii that have higher specific activity at lower temperatures. In a separate experiment, it was verified that there was a clear correlation between ortho-Nitrophenyl-β-galactoside (ONPG) concentration and the activity of enzyme beta-galactosidase (β-gal). Higher concentrations of ONPG yielded higher absorbance read by a photospectrometer at 421nm. Since Exercise 1 was conducted under standard atmospheric conditions of a standard classroom setting, further experimentation was profitable to determine whether or not temperature had a significant impact on enzyme activity or not.
The iodine test determines the presence of starch in biological materials. It is predicted that, if starch is not present, the solution with iodine remains yellow. However, if starch is present the solution with iodine becomes a blue-black colour. Plants have starch as the storage polysaccharide (glucose units held together by glycosidic bonds) while animals have the equivalent of glycogen. In this experiment, the dark blue colour is visible because of the helical amylose and amylopectin reacting with iodine (Travers et al., 2002).
Prior to the amylase extract being placed into the wells, the wells were prepared with 3 drops of iodine solution which works as an indicator of starch presence. We measured the presence of the starch with a 5 number color system. The lightest color, yellow, being number one and having the lowest concentration of starch, while the darkest color, a blue black,being number 5 and having the highest concentration of starch. The results showed that the optimal temperature for the bacterial amylase, Bacillus licheniformis, was 55℃, while the optimal temperature for the fungal amylase, Aspergillus oryzae, was of 25℃. The bacterial amylase, Bacillus licheniformis, had the
1.1 Abstract The purpose of quantitative analysis of protein using a spectrophotometer is to measure the concentration of proteins in a given sample. The experiment is conducted by laboratory method (Biuret Test) and using spectrophotometer to analyze the absorbance of reactants at 540 nm, hence determining the concentration of the proteins in a given sample. The purpose of stopped enzyme assay to study B-galactosidase is to determine the effect of temperature and concentrations of substrate on enzyme activity.
For example, maltose is the combination of two α-D-glucose which is found in germinating grain and is formed during the hydrolysis of starch to glucose during digestion. Lactose is the combination of β-D-galactose and α-D-glucose, it is digested by the enzyme lactase. Combination α-D-glucose with β-D-fructose will form a sucrose which it can be found in sugar cane and sugar beets. The invert sugar is produced by the hydrolysis of sucrose under acidic conditions, which break into glucose and fructose and it is sweeter than sucrose because of the present of fructose. This can be occur in the spread due to it contain acid-containing
Generally, the average amylase concentration was highest for those who reported having high ancestral starch diets and lower for the moderate levels. Although only one data point was reported for low ancestral starch diet, it still followed the trend of a decreasing amylase concentration with decreased starch in the ancestral
Amylase is the enzyme secreted by the oral cavity and can be found in the saliva glands. As soon as mechanical digestion begins, amylase digest the long, starch polysaccharide molecules found in food and breaks them down into smaller, simpler disaccharide molecules known as maltose. Maltose still needs to be digested further for absorption to take place in the small intestine. So, the enzyme maltase breaks maltose down into glucose. Other disaccharides are broken down by other carbohydrase enzymes.
The process of malting barley is a relatively simple process in which barley is
5 water bath were set up each to10 °C. (5 were used do the experiment faster) 5 cm3 of starch solution were added into the 5 test tubes that were labeled test tubes. Then 5 cm3 of amylase enzyme was added into the other 5 test tubes that were labeled. Put one of the starch solution test tube (preferably the one labeled 1) and one of the test tube containing amylase into the water bath (10 °C).
Life Cycle of a Seed Plant Annotated Bibliography Dante, R., Larkins, B., & Sabelli, P. (2014). Cell cycle control and seed development. Frontiers in Plant Science, 5. Dante et al. mention that seed development is complex and needs a coordination of the processes including metabolic, genetic, environmental cues and physiological pathways. According to the article, different cell cycle types often occur in sequential and in overlapping manner when the endosperm and embryo are developing.
Along with being found in plants, they are also present in liver cells, kidney cells, leukocytes and erythrocytes. For the concentration of enzyme experiment, the hypothesis was if the concentration of an enzyme increases, then the enzyme activity will increase as well. The hypothesis was proven to be true, because there are more enzymes to react with substrates. For the enzyme—factors affecting, the hypothesis concluded was if the temperature increases, than the enzyme activity will increase. This however was proven wrong, because enzymes become unstable at higher temperatures.