An Investigation into the activity of A Amylase

An Investigation into the application of A Amylasealpha amylase is an enzyme. It specific binds with water and starch . It hydrolyses starch and glycogen to give glucose and maltose. It acts on the alpha bonds of polysaccharides. Because of the specificity of the enzymes employment the structure of the enzyme must be precise. Any factor which will cause denaturation of the enzyme will restrict its rate of activity. Two of the most important factors effecting Alpha amylase activity atomic number 18 temperature and pH. The following experiment is designed to investigate the effect of different environmental temperatures and pH on the activity rate of barleycorn amylase.Materials and MethodsAs per schedule, No procedures were interpolated from original schedule.ResultsThe results be recorded as the time when colour change indicated that all the starch had been hydrolysed. A dark blue-black colour signify the presence of starch. When this colour is lost and an amber-yellow colour develops indicating that all the starch is hydrolysed, the time is recorded.The results indicate that the activity of alpha amylase increases with decreasing acidity and is highest at pH7. The trend in effect of temperature on amylase activity is that it increases in the middle range but is inactive at extreme temperatures. The results do not agree with the evaluate results of previous similar experiments and repeats of the same experiment within the class do not concur with othersDiscussionThe hypothesis being tested is that enzymatic reactions argon realized by a number of external factors. Temperature and pH are thought to be the most important extrinsic factors. The objective was to examine the activity of the enzyme -amylase under the effect of increasing environmental temperatures and increasing pH takes and to determine the optimal temperature and pH for Alpha amylase activity.The starch medium selected was Barley. This is a good choice imputable to its ready availabili ty, the ease of preparation and the frame of work available on understanding its germination process due to its importance in the brewing industry. Barley is composed of 53% to 65% dry weight starch (Fox et al (2003), MacGregor 1978, Sanford et al 2003). Barley produces its own amylases during the germination period. The changes in the levels of - amylase detected in barley during germination are outlined by McGregor et al (1984). The breakdown of starch in barley involves two types of amylase -amylase and -amylase. The former works by hydrolysing the 1-4 bonds within the glucose strand exposing non-reducing ends for the beta amylase to split (fig 3.) (Keusch 2003). Prior to germination there is no amylase detected in Barley, a trace was bring later 24 hours germination, but after that it was found to increase rapidly (MacGregor 1978).figure 3. www.uni-regensburg.de/Fakultaeten/nat_Fak_IV/Organische_Chemie/Didaktik/Keusch/Grafik/sucrose.gifimgrefurlThe optimal temperature and pH for -amylase extracted from barley is well studied. Fox et al (2003) state an optimal temperature of 65C and a pH of 5.5. ORourke (2002) gives optimal values of 67C and pH 5.2 (table 3) while lower temperature values of 55C for optimum activity of alpha amylase are given in other papers (Al-Bar 2009 and MacGregor 1978). The first two papers are written from an industrial and brewing viewpoint whereas the latter are written from a pure scientific evaluation of the characteristics of barley, this may have some bearing on the different temperatures cited keeping in mind that 60C is the temperature used in mashing.The optimal temperature for amylase activity differs for different sources, Azuki beans , finger millet and wheat have optimum temperatures of 70C, 45C and 55C independently (Al-Bar 2009). In mammals the temp ph of the body fluids are kept constant at homeostatic condition. If the body temp ph varies from the optimum body temp ph, the enzymes activity decreases and the ce llular respiration process which produces ATP energy cell mover would be affected in the body. Less ATP energy the body cells cannot perform the work which they need. The results of the amaze experiment did not comply with these expected results. From the literature the theory was that Amylase activity would increase with increasing temperature until a maximum level would be reached around 55C and there after it would decrease as it was denatured at higher temperatures, ultimately showing no activity at 80C. Likewise the effect of pH was anticipated to find increasing amylase activity with increased pH level to optimal between 5-6 and a decrease reaching neutral pH. While the average results were basically compliant there was a great variation in individual group results.Amylases are important across the spectrum of living organisms they are required for the breakdown of carbohydrates, which is one of the four essential food groups and the important source of energy to living cell s. Amylases are enzymes that increase the activity of a reaction without being consumed in the reaction. The reaction they are involved in is hydrolysis of carbohydrate which is the cleaving of bonds and the addition of water (Hogg 2005). Two main forms exist, alpha amylase and beta amylase, both hydrolyse carbohydrates but in different way. Alpha amylase is the faster acting as it can act on every part of the carbohydrate chain but beta amylase can only act on the non reducing ends which are produced in increasing amounts after the activity of alpha amylase. In plants amylases are largely involved in the germination of seed temperature may be the trigger for onset of this process. In animals amylases are found in association with the digestive system, ptyalin in the mouth and from the pancreas. The amylase enzymes hydrolysis the disaccharides sugar and maltose into monosaccharides glucose galactose which are the smaller molecules of starch that are suitable for absorption in the s mall intestine for ultimate body use. As an enzyme it binds the substrates together, promotes the hydrolysis and frees the products (fig 4.), and if denatured at high temperatures this cannot happenFigure 4. http//kvhs.nbed.nb.ca/gallant/biology/biology.htmlFrom the results obtained and the discussion hitherto the indications are that the results of the present experiment are in error. The anticipated optimal temperature and pH based on previous experiments were Temperature 55C and pH 5. The optimal results from the present experiment were 22C and pH 6.4. This is a well practiced experiment with well recognised results. looking at the results where some conform to the expected and others have results that are un expected it is most likely that the deviation is due to human error, as in not mixing solutions correctly, not maintaining the test tubes at a constant temperature, mixing up samples and not monitoring the time of reaction correctly. The observation of colour changes is also highly subjective, the precise time of change being difficult to detect. Improvements could include usinga spectrophotometer to measure change of amount of starch remaining and then it must be blanked correctly in the first place use.shaking equipment to ensure correct mixingusing the same amylase as preparation by different groups might not have been adequate absorption of substrate, exactness of measurement.Lack of closer attention by the operator can lead to error. Reaction rates, solutions and indicator need to be observed or measured correctly to avoid errors.In conclusion, to improve on the experiment in future, proper attention and observation is very important.