AustinPeayStateUniversityDepartmentofChemistry

CHEM1021

BREAKINGDOWNSTARCHUSINGSALIVARYAMYLASE
Caution: You will be using a Bunsen burner and glassware to create your own constant water bath.
Appropriate caution should be exercised when dealing with the Bunsen burner, hot water, and
glassware.
Purpose:Manyplantsstoretheirenergyintheformofstarch,apolysaccharidemadefromrepeating
unitsofthemonosaccharideglucose.Ourbodiesbreakdownstarchintotheindividualglucoseunits,
whicharefurthermetabolizedintoCO2andwaterthroughtheprocessofglycolysisthisistheprocess
we commonly call digestion. The enzyme amylase is present in our saliva and begins the process of
digestionwhenstarchcontainingfoodsenterourmouths.Inthislabyouwillmeasuretheamountof
amylasepresentinyoursalivabymonitoringthebreakdownofstarch.
Introduction:Youcanprobablynameavarietyoffoodsthatarereferredtoasstarchy.Suchfoods
include potatoes, rice, wheat, and corn. In essence, the starch is a long biopolymer made from
thousands of repeating
glucose units. When
these glucose units are
attached in a straight
line,thetypeofstarchis
called amylose. When
the units are branched,
the type of starch is
called
amylopectin.
Natural starch contains
both forms of the
polymer(Figure1).

Thelongchainsofglucosethatmakeamyloseformatighthelix.Ifwemixaniodinesolution
withtheamylose,theiodinewilllodgeitselfintheinteriorofthehelix.Thiscausesthesolutiontoturn
a dark blue color. If the enzyme amylase is added to the solution first, it will chemically remove one
glucosemonomeratatimefromtheends.Additionofiodinetothissolutiondoesnotgiveabluecolor
but will be colorless (Distinguish between clear and colorless; colorless means without color, whereas
clear means see through. Koolaid is clear, but is not colorless. Water is clear and colorless. Non
flavoredgelatiniscolorlessbutnotalwaysclear).

All enzymes speed up a reaction to a time scale useful for

our bodies. Starch can be broken down over thousands of years
withoutaidofotherchemicals,oritcanbebrokendowninlessthan
an hour with the enzyme amylase (Figure 2). Not everyones
amylaseworksatthesamerate.Thisiscalledtheactivityofthe
enzyme.Inthislabyouwillbedetermininghowwellyour(oryour
lab partners) amylase works by how fast it breaks down a starch
solution. You will report the activity in terms of the amylase

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CHEM1021

BREAKINGDOWNSTARCHUSINGSALIVARYAMYLASE
number,whichisthenumberofmillilitersofa1%starchsolutionthatcanbedigestedin30minutesby
1mLofsaliva.
Materials:
114intesttubesandrack

Ringstand

Ironring

Bunsenburner

10,25,and100mLgraduatedcylinder

Procedure:

50,150,and400mLbeakers
waxpencil
110oCthermometer
2%starchsolution
0.01Miodinesolution

1. Preparea37oCwaterbathbyheatingwateroveraBunsenburnerina400mLbeaker.Usethe
thermometertomonitorthereaction.Toohotortoocold,andtheenzymeswontwork.Goon
toStep2whileyouarewaitingforyourwaterbathtostabilize.

2. Making3amylasesolutions:Fillacleantesttubewithabout10mLofsaliva.Youwillbemaking
twodilutionsofthissolutionsoyouwillhavea1%,10%,and100%(undiluted)solution.Ina10
mLgraduatedcylinder,add1mLofsalivaand9mLofdistilledwaterforthe10%solution.Pour
thisintoaclean50mLbeakertomix.Ina100mLgraduatedcylinder,putin1mLofsalivaand
99mLofwaterforthe1%solution.Pourthisintoaclean150mLbeakertomix.

3. Setup10reactiontubes(labelwithawaxpencil)ofvaryingsalivaconcentrationsinatesttube
rackasfollows:
Tube#
Saliva
Distilledwater Finalsaliva%
1
3mL100%
0mL
100%
2
2mL100%
1mL
66.7%
3
1mL100%
2mL
33.3%
4
3mL10%
0mL
10%
5
2mL10%
1mL
6.7%
6
1mL10%
2mL
3.3%
7
3mL1%
0mL
1%
8
2mL1%
1mL
0.67%
9
1mL1%
2mL
0.33%
10
0mL
3mL
0%

4. Start the reactions: Add 3 mL of a 2% starch solution to the tubes. Mix, and then
simultaneouslyaddall10tubestothewaterbath.Afterexactly30minutes,add2dropsofthe
iodinesolutiontothetubesandrecordtheresultantcolor.A bluesolutionmeansstarchstill
remains,andacolorlesssolutionmeansallstarchhasbeenbrokendownintoglucose.

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CHEM1021

BREAKINGDOWNSTARCHUSINGSALIVARYAMYLASE
5. Determinetheamylasenumber:
a. Find the most dilute solution of saliva that still digested the starch (it didnt turn blue
uponiodineaddition).Thisistheleastamountofsalivathatcoulddigestthestarchin
thehalfhourtime.

Example: If you look at the test tubes and find that tubes 1 through 7 remained
colorless,and810werestillcolored(10shouldalwaysbecoloredsinceitiswithoutany
enzyme),thenweknowtube7,the1%salivasolution,wasthelimitonamountofsaliva
needed to digest the amylose. This is the tube we will use for the amylase number
calculations(determinationofactivity).

b. DeterminethenumberofmLofsalivainthetesttubeofinterest.
Example:Fortube7,therewas3mLofa1%salivasolution.TofindthemLofsalivain
thesample,multiplythepercentsolutionby3mL:
3
100

1% 3
100

0.03

c. Determine the amylase number. The amylase number is the number of mL of a 1%

starchsolutiondigestedpermLofsalivain30minutes.Becauseweused3mLofa2%
starchsolutioninsteadofa1%solution,wemustaccountforthatinourcalculations.A
2% solution of 3 mL is equivalent (in starch content) to 6 mL of a 1% solution. The
highertheamylasenumberis,thefaster(better)theenzymeworks.
Example:Fortube7:

1%

6
0.3

200

Waste Disposal and Cleanup: Dispose of solutions according to instructions provided by your lab
instructor. Wash all glassware thoroughly, rinse with deionized water and return to their original
places.

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CHEM1021

BREAKINGDOWNSTARCHUSINGSALIVARYAMYLASE

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CHEM1021

BREAKINGDOWNSTARCHUSINGSALIVARYAMYLASE

DATASHEET

Name:__________________________________

Partner:________________________________

1. Fillinthefollowingtable(spaceforcalculationsprovidedontheback):
Tube#
Saliva
Distilledwater Finalsaliva% Colorafteriodine TotalmLsaliva
1
3mL100%
0mL
100%

2
2mL100%
1mL
66.7%

3
1mL100%
2mL
33.3%

4
3mL10%
0mL
10%

5
2mL10%
1mL
6.7%

6
1mL10%
2mL
3.3%

7
3mL1%
0mL
1%

8
2mL1%
1mL
0.67%

9
1mL1%
2mL
0.33%

10
0mL
3mL
0%

2. Whichtubewillyouuseforcalculatingtheamylasenumber?____________________________

3. Amylasenumber(showyourcalculations):
Calculations:

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AustinPeayStateUniversityDepartmentofChemistry

CHEM1021

BREAKINGDOWNSTARCHUSINGSALIVARYAMYLASE

POSTLABQUESTIONS
Name: _________________________________________
Partner:__________________________________________

1.Whydidyouusea2%solution,whentheactivitydefinitionreliesona1%solution?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
2.Whydoesacrackertastesweetafterithasbeeninyourmouthforawhile?

3.Whatisthestructuraldifferencebetweenamyloseandamylopectin?Whatisthestructuralsimilarity
ofamylaseandamylopectin?

4.Ifonepersonsamylasenumberis200andanotherpersonsamylasenumberis150,whichperson
candigestlargeramountsofstarchmoreeffectively?

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AustinPeayStateUniversityDepartmentofChemistry

CHEM1021

BREAKINGDOWNSTARCHUSINGSALIVARYAMYLASE

PRELABASSIGNMENT

Name:_____________________________________

Partner:___________________________________

1. Whatisthepurposeofanenzyme(generically,notjustamylase)?
______________________________________________________________________________
______________________________________________________________________________
2. Whydoesthewaterbathneedtobeat37oC?
______________________________________________________________________________
______________________________________________________________________________
3. Iftube3isthemostdilutesolutionthatremainscolorlessuponiodineaddition,whatisthe
amylasenumber?Showyourwork.

4. Whatmonosaccharidewillbepresentinthetesttubeafterenzymaticdigestion?
_________________________

5. Inthislabweassumethattheamountofamylaseinsalivaisthesamefrompersontoperson.
Whyisthisimportanttoourcalculations?
_____________________________________________________________________________________
_____________________________________________________________________________________
______________________________________________________________________________

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