INTRODUCTION α-Amylases(E.C. 3.2.1.1.) are starch-degrading enzymes that catalyze the hydrolysis of internal a-1,4-O- -glycosidic bonds in polysaccharides with the retention of a-anomeric configuration in the products. Bacillus amyloliquefaciens find potential application in a number of industrial processes such as in food, fermentation, textiles and paper industries [1]. Most of the a-amylases are metalloenzymes, which require calcium ions (Ca2+) for their activity, structural integrity and stability[2].
The enzymatic hydrolysis is preferred to acid hydrolysis in starch processing industry due to a number of advantages such as specificity of the reaction, stability of the generated products, lower energy requirements and elimination of neutralization steps. Due to the increasing demand for these enzymes in various industries, there is enormous interest in developing enzymes with better properties such as raw starch degrading amylases suitable for industrial applications and their cost effective production techniques.
The enzymatic conversion of all starch includes: gelatinization, which involves the dissolution of starch granules, thereby forming a viscous suspension; liquefaction, which involves partial hydrolysis and loss in viscosity; and saccharification[3],involving the production of glucose and maltose via further hydrolysis.
The amylases of microorganisms have a broad spectrum of industrial applications as they are more stable than when prepared with plant and animal α- amylases [4]. Amylases are among the most important enzymes and are of great significance for biotechnology, constituting a class of industrial enzymes having approximately 25% of the world enzyme market [5]. They can be obtained from several sources, s...
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...derepressedBacillus subtilisKCC103 utilizing sugarcane bagasse hydrolysate. BioresourTechnol99: 3044-3050.
5. Bradford, M.M., 1976. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72: 248-254.
6. Sivaramakrishnan, S, . et al.: α-Amylases from Microbial Sources, Food Technol. Biotechnol. 44 (2) 173–184 (2006)
7. Vishwanathan .P.,Surlikar, N.R. (2001) Production of a-amylase with Aspergillusflavuson Amaranthusgrains by solid-state fermentation, J. Basic Microbiol. 41 57–64.
8. Haq, I., Ashraf, H., Qadeer, M.A., Iqbal, J. (2005) A source of alpha amylase production by Bacillus licheniformis, Bioresour. Technol. 96:1201–1204.
9. Pandey, A., Nigam, P., Soccol, C.R., Soccol, V.T., Singh, D., Mohan, R. (2000). Advances in microbial amylases. BiotechnolApplBiochem31:135-152.
Table 6 shows the results of the biochemical tests. The isolate can obtain its energy by means of aerobic respiration but not fermentation. In the Oxidation-Fermentation test, a yellow color change was produced only under both aerobic conditions, indicating that the EI can oxidize glucose to produce acidic products. In addition to glucose, the EI can also utilize lactose and sucrose, and this deduction is based on the fact that the color of the test medium broth changed to yellow in all three Phenol Red Broth tests. These results are further supported by the results of the Triple Sugar Iron Agar test. Although the EI does perform fermentation of these three carbohydrates, it appears that this bacterium cannot perform mixed acid fermentation nor 2,3-butanediol fermentation due to the lack of color change in Methyl Red and Vogues-Proskauer
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.
Moreover, the sensitivity and specificity of the western blot (Immunoblotting) enables it a common technique for determining specific protein levels in clinical samples. Since the antibody is specific to the antigen immunospecificity it enables the target protein to be identified. Western blotting can produce quantitative data about that protein, which in this case shows the difference between bands in each of the protein samples. The western blot is an analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. The proteins are then transferred to a membrane (in this case, nitrocellulose), where they are stained with antibodies specific to the target protein [1] [2].
called an active site. This active site is made by a few of the amino
enzyme. As I'm sure you are well aware, enzymes do not get used up in
In this investigation, the concentration of enzyme will be inversely proportional to the time taken for starch to be digested, until at a certain point where it will level out. It will level out because, all the substrates would have been used up, therefore there will be no more substrates for the enzymes to work on. In effect, the concentration of the substrate will act as a limiting factor. However, enzyme concentration will be directly proportional to the rate of reaction.
The artificial enzymes have a higher optimum temperature, (temperature at which they react fastest), and so also have a higher point of denaturisation. This means that the enzymes did not denature at 60 degrees Celsius as I predicted, so a reading at 80 degrees Celsius was taken. In this temperature the enzymes did denature as expected meaning that the reaction did not occur, so had the slowest rate of reaction possible. Evaluation ---------- I believe that the results recorded for the experiments undergone are reliable enough to base my conclusions upon.
Glycoside Hydrolases are classified into 108 families according with the amino acid sequence similarities. One of these families is GH1 (Glycoside Hydrolases 1), this family consists of enzymes with various substrate specificities, and the enzymes are present is bacteria, Archaea and Eukaryota. The 3D structure of 18 of these enzymes had been determined, and although the extent of sequence varies between 17% and 45%, all the enzymes have a common (β/α)8-barrel motif, and two catalytic glutamate residues located at the C-terminal end of β-strands 4 and 7, which may give a clue about the mechanism of these enzymes.
...e substances at 37.5̊C due to the fact that in the previous experiment, this was found to be the optimum temperature that catalase reacts at. It was because of this constant that I used the set of data of the catalase at 37.5̊C from the first experiment to provide a neutral environment for the experiment. The way in which the data was collected for the first experiment was identical to that needed to be done by the second. From this data, it was determined that the neutral environment for the catalase had the best results, which makes it clear that when the enzyme is in a pH of the opposite extremes such as basic or acidic, it is un able to function properly. When it is too basic then the enzyme will become inactive and when the enzyme is too acidic then the enzyme will denature, both rendering it unable to function at its optimum efficiency that all enzymes need.
at a volume of 4cm3. The preliminary work also proved to me that my basic method worked without any setbacks that may affect my results. Variables:.. The variables involved in the rate of reaction between amylase and starch are. The volume of amylase The volume of starch
Investigating the Effect of Enzyme Concentration on the Hydrolysis of Starch with Amylase Aim: Investigate the effect of enzyme concentration on the rate of an enzyme-controlled reaction. Using amylase and starch as my example. Introduction: I am investigating the effect of the concentration of the enzyme, amylase on the time taken for the enzyme to fully breakdown the substrate, starch to a sugar solution. The varied variable will be the concentration and all other variables are going to be fixed. The different concentrations will be: 0.5% 0.75% 1.0% 1.5% 2% An enzyme is a class of protein, which acts as a biological catalyst to speed up the rate of reaction with its substrates.
If I was to do this experiment again I might use a Fungi amylase to
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.
= Before conducting the experiment I would conduct a simple test for the protein by placing a sample of the albumen into a test tube and add biurett reagent. This contains copper (II) sulphate and sodium hydroxide.
An enzyme is a specialized protein that acts as a catalyst and facilitates complex metabolic processes. An enzyme, like any protein, is a polymer made up of a long chain of amino acids. The sequence of amino acids is determined by the DNA template in which it was made, and the amino acids are attached together by peptide bonds. Cross linking takes place between the R groups of the amino acids and forms a unique three dimensional molecules. The structure and spatial configuration of an enzyme, especially its binding site, is key to its optimal function and activity. This 3-dimensional structure can easily be altered by environment factors, such as salinity and pH. Each enzyme has a binding site in which chemical bonds are achieved with their