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ABSTRACT

The Effects of Varying Starch Concentration on a Solution of Amylase:

Measurement of Enzymatic Rate Changes Using IKI. Brooke Good Student,

Functional Biology, Section 1003, Southwest Texas State University, San Marcos, TX

78666

The relationship between starch concentration and the enzymatic rate of amylase were

investigated. Nine experimental assays were created all of which contained a solution of

IKI, starch, saliva, and pH buffer. The nine assays were set up under four separate

conditions containing a varying dilution of a 1 % starch concentration. The assays were

then timed and their IKI absorbencies were noted. The assays containing the greatest

percent starch concentration, 1%, had the highest enzymatic rate. As starch percentage

was decreased enzymatic rate decreased. It was concluded that starch concentration has a

direct effect on the enzymatic rate of amylase. The findings were consistent with

experiments of the past of this nature performed by early scientist.

INTRODUCTION

Enzymes are perhaps one of the most important proteins of the human body.

Enzymes such as amylase, an enzyme that breaks down carbohydrates, work by means of

surface catalysis. In other words, the surface of the enzyme enables other molecules to

react in a manner they would not be able to without the surface of the enzyme present.

Enzymes achieve this by lowering the amount of activation energy needed for anabolic

reactions, allowing these reactions to occur as catabolic reactions would. Enzymes are

generally large proteins made up of several hundred amino acids, and often contain a

non-proteinaceous group called the prosthetic group that is important in the actual

catalysis. In an enzyme-catalyzed reaction, the substance to be acted upon, orsubstrate,

binds to the active site of the enzyme. The enzyme and substrate are held together in an

enzyme-substrate complex by hydrophobic bonds, hydrogen bonds, and ionic bonds

(Nichols and Cholewiak, 1991).

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Enzymes are not only important because they keep the metabolic pathways free of

congestion but they are also key in digestion. Enzymes are needed to perform an infinite

number of tasks within the human cellular system. Without enzymes the body could not

possibly function properly, let alone sustain life. Enzyme productivity like many other

things in our body tend to vary based on outside factors. Environmental parameters such

as temperature, pH, and substrate concentration, such as starch, all cause changes in

enzyme productivity. How does a varying intake of starch affect the rate of enzymatic

catalysis in our body? Or does it? This will be the focus of this particular experiment.

Will varying starch concentrations directly or indirectly affect the rate of enzyme

catalysis, specifically amylase, an enzyme found in saliva? Starch is a polysaccharide

found in many of the foods eaten on a daily basis, thus its importance.

It might be assumed that starch concentration does not affect amylases ability to

catalyze. However, through the use of IKI, a solution of Iodine and Potassium Iodide

(optimal 660nm), which is commonly used as an indicator of starch concentration, we

will examine if starch concentration does in fact have an effect on the rate of enzyme

catalysis.

MATERIALS & METHODS

Dilutions of 1 % starch solution, 0.0 % (blank), 0.25 %, 0.50 %, and 0.75 % were

prepared for four controls, one for each starch concentration to be used as blanks. We set

the blanks up the same as the nine experimental cuvettes; each of which contain 1mL of

diluted starch solution and IKI, 1.2 mL saliva, 0.8 mL pH buffer, but without the IKI

making up the 4mL solution difference with an increase amount pH buffer. For the

experimental cuvettes we first pipet the starch solution and pH buffer into each of the

experimental cuvettes. Next we added saliva to all of the cuvettes and began timing. At

30-second intervals we added the IKI solution to each cuvette starting with an immediate

zero reading up to 4 minutes and measured each absorbency. At completion, we repeat

the previous steps as before only using 0.25 %, 0.50 %, and 0.75 % starch solution

respectively, keeping all substance and cuvettes at 37 Degrees Celsius throughout the

duration of the experiment.

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RESULTS

As the starch concentration was increased a higher reading was observed and then

a steady decline over the 4 min period (Figure 1) . As seen in Figure 2, 1.00 % was the

most optimal of the concentrations. At this point amylase catalysis rate was at its highest.

Figure 1. Absorption Readings of IKI in Solutions of Varying Starch. Absorbnce

(nm) measured over time for varying substrate concentrations (1.0 %, 0.75 %, 0.50 %,0.25 %). Illustrated with a regression of 95% confidence for slope.

Figure 2. Rate of amylase activity (slope) at varying starch concentrations.

.001

.002

.003

.004

.005

.006

.007

.008

.009

.01

.011

Rate

ofAmylaseActivity(abs/sec)

.2 .3 .4 .5 .6 .7 .8 .9 1 1.1

% conc. starch

- .2

0

.2

.4

.6

.8

1

1.2

1.4

1.6

absorbance

-25 25 75 125 175 225

time (sec)

0.25 % Starch Conc.

0.50% Starch Conc.

0.75 % Starch Conc.

1.00 % Starch Conc.

Absorbance plotted against Time

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DISCUSSION

Outside experimentation, the body perhaps is the greatest test of enzyme

adaptability. It uses enzymes in countless ways, and in countless conditions thus the

importance of understanding the effects of its environmental parameters. Uponcompletion of the experiment we found that when temperature and pH are held constant

the activity of an enzyme system is determined by the relative concentration of the

enzyme and its substrate, starch in this case. Specifically, we found 1% starch solution to

result in the highest rate of enzyme activity. If there is an excess of substrate, the rate of

catalysis is directly proportional to the enzyme concentration. If enzyme concentration is

kept constant, as it was in the following experiment, then the rate of reaction is directly

proportional to the amount of substrate present. Thus, an increase in substrate, starch,

causes an increase in enzyme catalysis. However, this is only up unto the point when all

enzyme molecules are utilized, or saturated. At this point the enzymes have reached their

optimal catalyzing rate and will no longer increase in rate of productivity. A much larger

experimental group would have been needed in order to find amylases optimal catalysis

condition, or saturation point.

Results from the experiment supported our hypothesis and support the results

from previous work on amylase activity but under different experimental conditions

(Jensen et al., 1997; Skrabanja & Tufvesson, 2000). However, it is not clear why there

was a precipitous decrease in enzyme activity at 0.075% starch. The inaccurate data

could have occurred as a result of imprecise measurements of the parts of the solution. Or

perhaps the time delay posed by the fact that time was not properly compensated when all

parts of the solution could not be added by one person simultaneously, or even the

mixtures of the IKI and starch solutions could have been slightly off. All of these errors

could have been reduced however through more careful planning and accuracy during

preparation.

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REFERENCES

Jensen, M. S. Jensen, S. K., and Jakobsen K. 1997. Development of digestive enzymes in

pigs with emphasis on lipolytic activity in the stomach and pancreas. Journal of

Animal Science. 75:437-445.Nichols, B.A.D. and Cholewiak L. B. . 1991. A quantitative enzyme study using simple

equipment. ABLE. 218:89-99.

Skrabanja, V. and Tufvesson, F. 2000. Digestibility of starch systems containing

amylose-glycerol monopalmitin complexes. ABLE. 34:131-139.