Enzymatic Activity of Salivary Amylase Formal Report

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Department of Biological Sciences, ollege of Science Department of Biological Sciences, ollege of Science Date submitted: 8 September 2015

Date submitted: 8 September 2015

*katrinalee_gagan@yahoo.com *katrinalee_gagan@yahoo.com

!bstract !bstract

Salivary amylase, found in human saliva, is

Salivary amylase, found in human saliva, is an enzyme used to hydrolyze starch molecules.an enzyme used to hydrolyze starch molecules. Its enzymatic activity is affected by several factors, such as temperature and pH. The rates of  Its enzymatic activity is affected by several factors, such as temperature and pH. The rates of  enzy

enzymatimatic c actactivity of ivity of salisalivary amylase in vary amylase in diffdifferenerent t temptemperateratures and ures and pH pH were measurewere measured.d. Optimum temperature for the enzymatic activity of salivary amylase ranges from 3!" to 3#!" Optimum temperature for the enzymatic activity of salivary amylase ranges from 3!" to 3#!" and its optimum pH ranges from $ to #.

and its optimum pH ranges from $ to #.

"ntroduction "ntroduction  %n enzyme

 %n enzyme is a is a protein molecule that protein molecule that is is a a biologicabiological l catalyscatalyst t with three with three charactecharacteristics.ristics. &irst, the basic function of an enzyme is to increase the rate of a reaction. Second, most &irst, the basic function of an enzyme is to increase the rate of a reaction. Second, most enzymes act specifically with only one reactant, called a substrate, to produce products. enzymes act specifically with only one reactant, called a substrate, to produce products. The third and most remar'able characteristic is that enzymes are regulated from a state The third and most remar'able characteristic is that enzymes are regulated from a state of low activity to high activity and vice versa. The activity of enzymes is strongly affected of low activity to high activity and vice versa. The activity of enzymes is strongly affected by changes in pH and temperature. (ach enzyme wor's best at a certain pH and by changes in pH and temperature. (ach enzyme wor's best at a certain pH and te

tempemperatratureure, , its its acactivtivity ity decdecrereasiasing ng at at vavaluelues s ababovove e anand d bebelow low ththat at popoinint t dudue e toto denaturation. &or enzymes, denaturation can be defined as the loss of enough structure denaturation. &or enzymes, denaturation can be defined as the loss of enough structure rendering the enzyme inactive. This is not surprising considering the importance of  rendering the enzyme inactive. This is not surprising considering the importance of 

#n$%matic !cti&it% of Sali&ar%

#n$%matic !cti&it% of Sali&ar%

!m%lase

!m%lase

CHEMISTR CHEMISTR  Y   Y 

60

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tertiary structure in enzyme function and noncovalent forces in determining the shape of  enzymes.

Salivary amylase is the enzyme produced by the salivary glands. &ormerly 'nown as ptyalin, it brea's down starch into maltose and isomaltose. %mylase, li'e other  enzymes, wor's as a catalyst. %ll catalysts are enzymes, but not all enzymes are catalysts. % catalyst is a substance that hastens a chemical reaction but does not become part of the end product. %mylase digests starch by catalyzing hydrolysis, which is splitting by the addition of a water molecule. The presence and absence of starch can be confirmed by several tests such as the iodine test, )enedict*s and &ehling*s test. In general, a blue+blac' color indicates the presence of starch.

In the eperiment, the enzymatic activity and specificity of salivary amylase was eamined depending on changes in pH and temperature. &actors such as narrow range of pH values and different temperatures at which the enzyme ehibits its optimum activity were done.

'esults and discussion !( #ffect of )emperature

(ach enzyme has an optimum temperature at which it performs best. )elow or above this temperature, the enzyme loses its functionality. The Table below shows the results obtained on how enzyme activity of salivary amylase is affected by temperature.

)emp(  )ime +min( -.t +min/-

0 - 

12 // mins. .00

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25 - 

45 - 

The graph below shows the reciprocal of time against Temperature based on the data from Table above.

5 10 15 20 25 30 35 40 45 50 55 60 65 70 0 1 2

Chart Title

Column1

6lot of the reciprocal of time against temperature for the en$%matic acti&it% of sali&ar% am%lase

The graph produced a mountain li'e curve with the highest pea' indicating the optimum temperature for enzymatic activity. %t 3#!", enzymatic reaction of salivary amylase occurs slowly or not at all due to lac' of energy and heat. %s the temperature increases, its enzymatic also increases up until the optimum temperature. The graph shows that the optimum temperature of salivary amylase was 3#!".This applies to the human body since salivary amylase is suitable to function within these temperatures. %fter 3#!", the graph then steeply declines as a result of loss of activity. %t 1!" and #!", salivary

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amylase is denatured. The molecular conformation of the enzyme becomes altered as the hydrogen bonds responsible for its secondary, tertiary and 2uaternary structures are bro'en.

B( #ffect of p7

ost enzymes are active only over a narrow pH range and have an optimal pH, at which reaction is the fastest. %n increase or decrease in pH also causes denaturation in enzymes, thereby affecting their activity. The table below shows the results obtained on how enzyme activity of salivary amylase is affected by pH.

p7 )ime +min( -.t +min/-

0 - 

8(4 / min. / min.

9 - 

-5 - 

 The graph below shows the reciprocal of time against pH based on the data from Table above.

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1 2 3 4 5 6 7  ! 10 0 0"2 0"4 0"6 0" 1 1"2

Chart Title

#eries 3

6lot of the reciprocal of time against p7 for the en$%matic acti&it% of sali&ar% am%lase

The graph produced a mountain li'e curve with the highest pea' indicating the optimum pH for enzymatic activity at pH $ the graph was at its highest pea'. %t pH 4 5 /, salivary amylase is denatured due to high al'alinity which is ideal. %s pH increases, certain amino acids such as lysine and arginine are deprotonated, causing them to lose their net positive charge which also results to enzyme denaturation.

The activity of enzymes may be mar'edly changed by any alteration in pH, which in turn, alters electrical charges on the enzyme. "hanges in charge affect the ionic bonds that contribute to the enzymes tertiary and 2uaternary structure, thereby changing the proteins conformation and activity. Thus, pH+activity relationship of enzymes is dependent on the amino acid side chains present in the enzyme.

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Several factors affect the activity of enzymes. %mong these are the temperature and pH.  %t optimum levels of these factors, enzymes perform their function best. Optimum

temperature and pH differ from one enzyme to another. Salivary amylase is an enzyme found in human saliva which functions to brea' down starch to simpler compounds. Through the eperiment, it was found out that the optimum temperature of salivary amylase ranges up to 3#!" and its optimum pH ranges from 1+$.

#:perimental methodolog%

This procedure was for the determination of the optimum temperature of the amylase, where we put  ml of the enzyme solution in a large test tube and labeled it as 67". Then in a separate large test tube we added  ml of the buffered starch solution and incubated both test tubes for / mins. in an ice bath. 8et, we immediately mied and 2uic'ly too' three drops of the miture and simultaneously added two drops of the iodine solution onto a spot plate 9first plate: which then was our zero minute. %fter one minute interval, we too' three drops again of the miture and simultaneously added two drops of the iodine solution onto the second plate which then was our one minute. ;e repeated step 1 until a light yellow colored solution was observed and noted the time.

This procedure was for the determination of the optimum pH of the amylase, where we mied / ml of acetate buffer 9pH 6: and / ml < un buffered starch in a large test tube. Then in a separate large test tube we added  ml of  the enzyme solution. 8et, we incubated both test tubes for / mins. in a 3#7" water  bath. Then, we immediately mied and 2uic'ly too' three drops of the miture and

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simultaneously added two drops of the iodine solution onto a spot plate 9first well: which then was our zero minute. =astly, after one minute interval, we too' three drops again of  the miture and simultaneously added two drops of the iodine solution onto the second well which then was our one minute. ;e repeated the last step until a light yellow colored solution was observed and noted the time.

'eferences

9/: (nzymes. 9//:. >etrieved on Sept. 1, /1 from http?@@users.rcn.com@A'imball.ma.ultranet@)iologyBages@(@(nzymes.htmlCpHan dTemp

9: >ole of (nzymes in )iochemical >eactions. 93:. >etrieved on Sept. 1,/1 from http?@@www.elmhurst.edu@Dchm@vchemboo'@1#enzymes.html 93: Sethi, >. 90:. )iology. >achna Sagar Bvt. =td.? 8ew Eelhi

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