The intent of experiment is to detect amylase enzyme in different environment and detect of each environment by assisting coloring material alterations. Enzymes are biological molecules that catalyze many different chemical reactions. With few exclusions, all enzymes are proteins and each enzyme is specific to a certain chemical reaction. Enzymes must keep a specific three dimensional construction in order to work decently. If an enzyme ‘s construction is altered ( by heat or rough chemicals ) it may non work at all. This dislocation ( denaturation ) of an enzyme ‘s construction may be fatal
Amylase Enzyme
Amylase, which is normally found in spit and shooting seeds. It catalyzes the dislocation of amylum. When amylase reacts with amylum, it cuts off the disaccharide malt sugar ( two glucose molecules linked together ) . As the reaction progresses, less amylum will be present and more sugar ( malt sugar ) will be present.The activity of amylase can be observed by utilizing iodine.Because I reacts with amylum to organize a dark brown/purple colour. As amylase interruptions down amylum, less and less amylum will be present and the colour of the solution ( if I is added ) will go lighter and lighter. The colour alteration was observed utilizing spot-plates as illustrated on the diagram below.
Amylase activity was observed under four different interventions:
consequence of temperature
consequence of pH
consequence of substrate concentration
consequence of enzyme concentration
The Effects Of Temperature
Amylase is an of import metabolic enzyme. Its map is to catalyse the hydrolysis of amylum into glucose. At high temperatures, Amylase becomes denatured, denatured amylase no longer catalyzes the hydrolysis of amylum into glucose.
Consequence OF pH:
Based on these consequences, what is the optimum pH for amylase? Is this optimum pH considered acidic, basic/alkaline, or impersonal? Why does the activity lessening when the pH is excessively low or excessively high?
Apparatus
-Starch
-Amylase Enzyme
-KH2P04
-Na2HP04
-HCI
-Heater
-Beaker
-Falcon tubing
-Spectrophotometer
-Iodine
Procedure
1.0.27 g KH2P04 buffer solution PH 5 was prepared with 20ml
2.0.27g KH2P04 PH6 was prepared with 20ml
3.0.27g KH2P04 PH7 was prepared with 100ml
4.0.282g Na2HPO4 PH8 was prepared with 20ml
5.0.282g Na2HP04 PH9 was prepared with 20ml
6.20g Starch was besides prepared with 50ml cold H2O
7. To prove amylase activity with PH difference,5ml amylum,5ml buffer ( PH5,6,7,8,9 is used each ) and 1ml amylase were assorted each other.
8.10min later,0.5ml prepared sample was put into 5ml HCI.
9.At 620nm, the consequences were measured at spectrophotometer.
10. Second portion temperature effect,5ml starch,5ml PH7 buffer and 1ml amylase were assorted.
11.Prepared sample was put into different temperature 30,50,70 and 90C.
12.10 min later,5ml HCI was put into 0.5 milliliter prepared sample.
13.2-3 min later,5ml I was added into 0.5ml new sample
14.Absorbance of each was measured at spectrophotometer.
Observation
In this experiment, we tried to make different environment to analyze amylase enzyme activity.The environment differences could be provided by PH differences.Therefore we prepared different medium besides different pHs.K2.The graph was gained f & A ; Auml ; ±om our results.One of them is a graph that related to amylase activity at different PH.The other one is rela ted to amylase activity at different temperatures at changeless PH.With K2HPO4 PH 5.6and 7 were prepared and with Na2PO4 8and 9.Each readying process was applied.5ml amylum,5ml buffer,1ml amylase were added each other and so waited 10 min.After 10min,5ml HCI was added into 0.5 ml sample mixture.In a same manner, the mixture for temperature observation was prepared pH 7.And added I to stop of process. Optical density consequences were taken from spectrophotometry.This measuring was at 620nm.
pH buffer sample with amylase
0.074
0.027
0.026
0.043
0.074
Harmonizing to the consequences,
The smallest one can be think as a best one.How much enzyme is used is more indispensable point.If it is less one, it means amylum can non be used adequately.High amylum sum means that complex sum is besides high.The opposite 1 shows best activity amylase at smallest concentration.The coloring material is more light, smaller optical density could be think as best amylase activity.
Temperature sample with amylase
0.064
0.006
0.192
0.130
At 30C the coloring material is somewhat orangish.
At 50C the coloring material is excess visible radiation like iodine coloring material.
At 70C the coloring material is somewhat violet.
At 90C the coloring material is more violet than at 30C one like orange-purple.At changeless PH, the little concentration, at 50C.Because little optical density formed by little complex.It means that sum of amylum was decreased also.Best activity is 50C at changeless PH.
Consequence
Our purpose is to be related to activity of amylase.To detect it, we prepared different PH from KHP04 and Na2HP04 by adding acid or base. Usage both of them is related to interval of buffer.After readying buffer, we measure optical density at spectrophotometry.At different PH optical density spring besides different concentration.If amylase enzyme concentration with sample is little, it means enzyme is used complex is more little so activity of ezyme is best one in there.At different PHs, smallest concentration is at PH 7.And so we did 2nd portion of experiment by utilizing PH7.The chosen of PH7 is related to observation best amylase activity at first part.At PH7 we took sample with amylase enzyme concentration at different PHs.The smallest concentration is at 50C in 2nd part.The concentration is 0.006.The coloring material is more light like iodine colour.Starch is used with amylase and hence complex coloring material is more light also.The amylase enzyme activity is best one at 50C.This measuring is done at 620nm.
DISCUSSION AND CONCLUSION
Why is measured at 620nm? Why HCI is used for readying? What does Light colour mean? How does more heat affect rxn? During experiment, we want to distinct intent of experiment by replying these question.In this experiment, we related to consequence of different buffer and temperature.We prepared buffers at different PH.KH2P04 was prepared for PH 5,6,7and Na2HP04 for 8and 9.In first portion, at changeless temperature ( room temperature ) sample with amylase concentration was measured.At PH 7, we measured the smallest one.Small concentration means less complex less amylum and enzyme is used enzyme activity is high.Our consequence from measuring at PH 7 is 0.026.As a 2nd portion, changeless PH, temperature was changed and so observed the consequence of it.At 50 C, smallest optical density ( 0.0060 ) was found and the coloring material was excess light.It means more less complex there.In this experiment, I is used to observe amylum molecules by detecting colour change.Iodine and amylum were combined and so formed complex.The another point is why HCI is used.The acerb stops the enzymatic reaction and iodine reacts with amylum to bring forth bluish color.Activity of enzyme is besides essential.It can be used for denaturation detection.Starch reacts with I which is xanthous to organize bluish compound Amax=620nm.The strength of the bluish colour can be quantified spectrophotometrically by mensurating its optical density at 620nm.
