EFFECTS OF TEMPERATURE IN INVERTASE ACTIVITY Redentor D.L. Laureta III, Bea Czarina T. Loque, Lara Mae S. Lorenzo, Jeanette T. Lusung and Tobias Manuel Y. Maningat Grou Group p 5 2H 2H Med Medic ical al Tec Techn hnol olog ogy y Bioc Bioche hemi mist stry ry Labo Labora rato tory ry
ABSTRACT Several factors affect enzyme reaction rates, one of which is temperature. The aim of the experiment is to find the relationship between temperature and enzyme reaction rate, with the enzyme invertase. Invertase is a hydrolase which which reacts reacts with sucrose sucrose to form glucose glucose and fructose. fructose. Several Several test tubes with equal equal amounts amounts of sucrose sucrose and invertase when subjected to varying temperatures, and then DNS reagent was added. Each test tube was subjected to 95°C to form the ANS, characterized by a red-brown color, then analyzed with the spectrophotometer at 540 nm.
INTRODUCTION Enzymes are protein catalysts found in nature that speed up biological biological functions functions and processes processes that hat wou would othe otherw rwis ise e take ake a long long tim time to comple completel tely y finish finish.. The effici efficienc ency y of enzym enzymes es is affected by a variety of factors, most notably the temper temperatu ature re of the surro surround unding ing enviro environme nment. nt. Like pH, an extremely high or low temperature will also reduce the enzyme activity. The enzyme that will be used in this experiment is invertase, an enzyme found in plants and acts as a catalyst for the hydrolysi hydrolysis s of sucrose. Invertase Invertase will be obtained obtained through through extractio extraction n from baker’s yeast which ich will ill be done one in the the firs irst part part of the the experiment. The objective of the experiment is to observe and take note the optimum temperature in which enzymes react and process products, in this case, the optimu optimum m temper temperatu ature re of invert invertase ase and its reacti reaction on with with sucros sucrose, e, compar compared ed with with a blank blank control in the form of denatured e nzymes. This This expe experi rime ment nt also also invo involv lves es usin using g spectrophotometry to view the results of enzyme activity in different temperatures.
EXPERIMENTAL A. Materials and Compounds Used A. Materi Materials als and and Compou Compounds nds Used Used For extraction of invertase from yeast: 0.25g Baker’s yeast, distilled water For the effect of temperature on invertase activity: activity: 100mg/L 100mg/L Sucrose Sucrose standar standard d solution, solution, concentrated hydrochloric acid, 0.5M Potassium Hydroxide, Dinitrosali alicylic lic aci acid (DNS) NS) reagen reagent,1 t,10g/ 0g/L L Sucro Sucrose se sol soluti ution, on, 0.1M 0.1M buffer buffer solutions(pH 5), test tubes, pipettes, beakers, volumetric flasks, paraffin film, hot plate, UVVis spectrophotometer. B. Proc Proce edure dure 1. Extraction Extraction of Invert Invertase ase from Yeast In the act of drawing out the invertase, 0.25g of Baker’s yeast was weighed and added with
distilled water until a volume of 250 mL was achieved. The solution was allowed to stand for 20 minu minute tes s at room room temp temper erat atur ure e and and the the collection of the supernatant was followed upon the occurrence of sedimentatio tion. The supe supern rnat atan antt was was then then used used as the the enzy enzyme me stock solution for the other experiments. 2. Preparation of Denatured Invertase Stock Solution One hundred mL of enzyme stock solution was incubated in a boiling water bath for 10 minu minute tes. s. The The solu soluti tion on was was cool cooled ed and and the the supe supern rnat atan antt was was collec collecte ted. d. The The coll collec ecte ted d supernatan supernatantt served served as the denatured enzyme stock solution. 3. Determining the Effect of Temperature on Invertase Activity A 20, 30, 50, 60, 70 and 90 degrees Celsius water baths was prepared. Six test tubes with each each contai containin ning g 1.5 mL sucros sucrose e solut solution ion was prep prepar ared ed and and incu incuba bate ted d sepa separa rate tely ly for for 5 minute minutes s in each each water water bath. bath. In anothe anotherr test test tube tube,, 0.80 0.80 mL enzy enzyme me stoc stock k solu soluti tion on was was mixed with 19.20 mL 0.1M buffer solution with a pH of 5. Afterwards, 3 mL of dilute enzyme solution was added to all test tubes and were all all incu incuba bate ted d for for anot anothe herr 5 minu minute tes. s. DNS DNS reagent with amount of 3 mL was also added to all all tes test tube tubes s. The The tes test tube tubes s were were then then immersed in 95° 95°C water bath for 10 minutes for the development of the characteristic redbrown brown col color. or. The sol soluti utions ons were were coo cooled led and cove covere red d to prev preven entt evap evapor orat atio ion. n. The The same same procedures were used in the case of denatured enzyme. The absorbance was measured at 540 nm. Finally, the amount of sucrose hydrolyzed using sucrose standard curve was determined. The construction of sucrose standard curve was construc constructed ted in the dinitrosal dinitrosalicyli icylic c colorimetr colorimetric ic method.
RESULTS AND DISCUSSION 1
After spectrophotometry, the results were noted down, (Table 1.) and graphed (Fig.4.1) Table 1. Effects of Temperature on Invertase Activity Test Tube 1 2 3 4 5 6
Temperature 20 30 50 60 70 90
Absorbance 0.123 0.234 0.456 1.234 0.890 0.432
Fig.4.1 Graphical Presentation Activity Versus Temperature.
of
Invertase
Campbell, M.K., and Farrell, S.O. (2006). Biochemistry, Fifth Edition. California, USA: Thomson Brooks/Cole. Delvin, T.M., (2002). Textbook of Biochemistry with Clinical Correlations. New York, USA: WileyLiss. From the Internet Li, T.S. Effect of temperature on soluble invertase activity, and glucose, fructose and sucrose status of onion bulbs (Allium cepa) in store. (2004) Retrieved from http://informahealthcare.com/doi/abs/10.1080/0 9637480412331290512. 2/3/2011 Effects of pH (Introduction to Enzymes). (n.d.) Retrieved from http://www.worthingtonbiochem.com/introBiochem/tempEffects.html. 2/3/2011 Effect of High Temperature on Sucrose Content and Sucrose Cleaving Enzyme Activity in Rice Grain During the Filling Stage. (2006) Retrieved from http://www.ricescience.org/qikan/manage/wenzh ang/E060309.pdf . 2/3/2011 The Effect Of pH On Invertase Activity. (2003) Retrieved from http://www.coursework.info/ AS_and_A_Level/Biology/Molecules__Cells/The_E ffect_Of_pH_On_Invertase_Activity_L107500.ht ml
As noticed in the graph shown at Fig4.1, there is peak absorbance at 60°C. This point is the optimum temperature, where in the enzyme is most active. As the temperature increases, the rate of reaction also increases, similar to inorganic reactions. But as seen in Fig.4.1, at a certain temperature, the rate of reaction drops drastically, represented by the bell-shaped curve apparent in the graph. This phenomenon can be explained by the nature of the enzymes which are proteins. All proteins with a tertiary structure, like invertase, denature when exposed to enough high temperatures. Denatured proteins do not react as much as the normal proteins, therefore lessening the reaction rate of the entire system.
REFERENCES From Books Boyer, R. F. (2006). Concepts in Biochemistry (3rd ed.). Hoboken, NJ: Wiley.
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