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Experiment: enzyme Activity

Objetives:To be able to hydrolyze starch with the help of enzymes in the samplesTo identify prescen of enzymes in the samples y means of qualitative screening

aterials :!" test tubesTest tube rac#Tripod$unsen burner %asserole

&ire gauze$ea#er 'raduated cylinder (ipete

 Aspirator &ooden splint

)eagents*aliva+ugols solution

*trach solutionO,!- -a%l

.,./ 0%l%opper sulfate

1istiled water 

ethodology

The students first perpared all needed materials and reagents within the studentsvicinity for an orderly conduction of the experimintation and to lessen theprobability of human error,

The experiment was divided into " parts, 2irst the students prepared the catalseenzyme by grating a potato with the potato s#in, /. ml of water was then

communicated to the potato in a bea#er which was subjected to !. minutes withoccassional stirring, The sample was then filtered where the remants werethrown away by the students, The filtrate was then #ept for qualitative testing,

/ml of the catalase sample was mixed with hydrogen peroxide, The studentsimmediatley inserted a glowing wooden splint,

The students then conducted the part $ of the first part of the experiment wherein / ml of the sample was added with / ml of 3 moles of coodium hyroxide, Thestudents then mixed the sample by tapping the tewsttube with the palms of thehand, / drops of copper sulfate was then communicated to the mixture,

2or the second half of the experiment the students prepared the sample bycllecting !.4!/ ml of saliva from the students, The students then labeled 3 testtubes where all of the 3 test tubes contained !. ml of starch colution5 !ml of .,! -a cl solution5 and " ml saliva, Test tube ! has an addition of !ml of .,./ -0%l5 test tuibe " has an addition og ! ml of distilled water5 while test tube 3 hasan addition of ! ml .,.3 - .,./ -aO0,

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The students then mixed all of the test tubes and subjected the samples to a 36%water bath where in the students added " drops of lugols solution every 3 minuteinterval, The students stopped the addition where in the solution turns blac#,

+astly for the last part of the experimentation5 the students added !. ml of the

starch solution5 ! ml of .,! of the sodium chloride solution, The students thensubject test tube ! into a bea#er of room temperature water5 test tube " to a 36degree water bath5 and test tube 3 to a boiling water bath, the students thencommunicated " ml of the saliva sample onto the test tubes and was mixed,

78 )esults and discussion

1uring the first part of the experimentation the students isolated the ctalaaseenzyme residue which was thrown from the sample potato, The studentsacquired the said enzyme by means of grating the sample into a fine pulp whichwas then mixed occasionally with /. ml water, The *tudents then filtered the

enzyme where as it was acquired through the filtrate and not the residue whichwas not utilized in the experiment, The following observations were made whenthe filtrate was subjected to qualitative testing with the correlation on what theimplications are on a molecular level,

*ample9reagent observation

%ataalse extract9 hydrogen peroxide 7nstant formation of a large foamysubstance,

7n the first part of the experiment where as the students tested the presence ofcatalase from the sample5 /ml of the filtrate was added with / ml of / hydrogen

peroxide, *pontaneuosly a glowing wooden splint was communicated to themixture, The students observed a lage formation of foam rose at instant upon thecommunication of the glowing wooden splint,

The formation of the foam from the mixture concludes a positive result where asthe enzyme catalase reacted with the hydrogen peroxide, According to education,com ;".!3<5 %atalase acts as the catalyzing enzyme in the decomposition ofhydrogen peroxide,The statement above bac#s up the reaction that happened inthe test tube, &hen catalase is communicated with hydrogen peroxide ; itssubstrate< and is activated by the catalyst of heat :by means of the experimentthe glowing stic#5 catalase speed up the principle of decomposition to brea#

down hydrogen peroxide to water and oxide, The chemical equation belowilustrates the reaction given off by hydrogen peroxide and the enzyme catalase,

sample Observation

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%ataase extract9 3 -aO09%u*O= A blac# suspended preciptate matrixedin the solution

1uring the second art of the !st half of the experiment5 the nature of enzymeswas screened by the students by the addition of /ml og the catalase sample to /ml of 3 moles of sodium hydroxide> which was then shoo#, / drops of copper

sulfate was then added to the test tube with the mixture, The table above denotesthe observations made by the students from the said experiment,

%atalase is very sensitive to inhibition, Even the substrate ;0"O"<5 especially inhigher concentrations5 results in the enzyme inactivation,*ulphuric acidimmediately inactivates catalase and stops the reaction of 0"O" degradation, ;?niversity of assechussets $oston< according to the statement above sincecatalase a very sensitive enzyme5 addition of the copper sulfate will inhibit theenzymes activity from the reaction, The students observed in the experiment bythe addition of the copper sulfate to the solution5 a blac# precipitate form this isdue to the binding of the sulfuric acid to the catalase which caused the formation

of blac# precipitate meaning that the reagent is inhibiting the function of catalase,

1uring the second half part A of the experiment the students then tested thefactors that affect enzyme activity specifically temperature, The students used thesaliva samples and added varrious reagents which was then heated in acontrolled 36% water bath and analyzed when the solution would start todegraded, The table below denotes the reagents and time spent in order for thesolutions to be gegraded,

*amples 9differentiatedreagent

Time elapsed 1rops of +ugols

Test tube !9!ml .,./-

0%+

!@ minutes !" drops

Test tube "9 !l ditsilled0"O

!/ minutes !. dops

Test tube 39!ml .,./ --aO0

. minutes .

The addition of +ugols solution every 3 minutes was to test whether the samplestarch was completely degraded which only came a positive result only if the testtube turns purple to a bac# hue, Test tube ! garnered the longest time which was!@ minutes until starch was hydrolyzed with !" drops in order to confirm, Testtube " garnered only !/ minutes and was confirmed after !. drops, And astly test

tube 3 didnt need any time because even before heating the students noticedthat the starch was already hydrolyzed but to ensure it was still subjected to thetesting for !/ minutes with " drops in 3 minutes interval,

The reason why test tube 3 was sontaneuosly hydrolyzed was due to the addedreagent which was sodium hydroxide, *odium hydroxide reacted with the starchwhich caused al#aline hydrolysis, Alkaline hydrolysis is a simple, natural process by

which complex molecules are broken down into their constituent building blocks by the

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insertion of ions of water (H2O), H+, and OH between the atoms of the bonds that held

those building bocks together (alnmag,2!"!)# $here was the water ion is present in the

sodium hydroxide and hyrolusis the glycosidic bond which contain oxygen# %est tube no2 laso used alkaline hydrolysis but since water is only baseline alkaline, the amylase

 present in the sali&a had a more contribution

%est tube " took the longest because H' denatures the sali&ary amylase present in the

test tube cancelling out the reaction# %he principle on what hydrolyed the starch is due to

the water present in the starch solution and also degredation due to heat#

%est tube no# %ype of heat sub*ected time rops of

lugols

" oom temp water bath until end

time ofexperimenta

tion

"! drops +

until end ofexperiment

2 -.' water bath "/ mins "! drops- 0oiling water bath "2 mins 1 drops

%he students used the same reagent controls in the experiment but differentiated the type

of temperatures where in they will be sub*ected at# All of the samples contanied the starchsolution, a'l, and the sali&a sample# %est tube " was subected to a room temp water

 bath where in the students added 2 drops of the lugols solution until the experimentation

ended3 this is due to the fact that the amylase hasnt been reacted with the starch sampledue to the lack with the help of any heat# %he students obser&ed that there may be a

definite time frame where in starch will be hydrolyed in that kind of en&iroment but due

to the lack of time, the students ha&e not figured or obser&ed the definit time#

%est tube 2 was sub*ected onto a regulated -. ' water bath where in it took "/ mins and

"! drops of lugols solution in order for the oncifrmation# %he results were similir to thetime frame obser&ed in the pre&ious part of the experimentation# %hus may be due to the

similar heat communicated to the substances#

astly for the third test tube, a boiling water bath was communicated to the sample# 4ttook the sample "2 mins with the help of 1 drops of the lugols solution# ue to the high

heat added, it helped catalyed the solution in hydrolysis of starch with the amylase# 4f

more heat and a longer period of time was to be obser&ed amylase would also ha&eundergo degradation#

According to chem blogspot(2!"/)ugol5s iodine yields a blueblack color in the presence of starch# %he statement abo&e shows the use of the lugols solution in the 2 parts

of the last half ot he experimentation# %he iodine would stick into the chains of the starch

creating a blueblack complex# 4f when the tested sample would create this reaction, it

means that the strach is still not hydrolyed as per the experiment# 4f the solution wouldgi&e *ust a black hue it would mean a positi&e result for which the starch would be

hydrolyed and the glycosidic chains are broken# %he figure below denotes the

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illustration on how the mechanism of lugols solution works

 Accorfing tobbc; ".!.< the use

of heat in thereaction helps theenzyme achieve itsmaximumpotential, This wasobserved in part 3of the experimentfor which the boiling water bath garnered the fastest resulting time in whichstarch was hydrolyzed, The heat helped the amylase speed up the reaction, 7-collinear with this the test tube ! got very ngative results because only roomtemp was used as the heat source,

)eferences http:www,education,compdfactivatorhttp:www,umb,edu,plphotopli#i&+Bjednost#iza#ladBbiochemiiBle#ars#iejpdfbiochemistryBwor#boo#,pdf http:www,alnmag,comarticles"..=.@al#aline4hydrolysis4processhttp:generalchemistrylab,blogspot,com".!!!"iodine4test4for4starch4and4glycogen,htmlhttp:www,bbc,co,u#schoolsgcsebitesizescienceaddBaqaproteinsproteinsrev3,shtml