International University School of Biotechnology

Vietnam National University – HCMC Department of Biochemistry

ENZYMOLOGY LABORATORY
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REPORT 3

EFFECT OF ENZYME
CONCENTRATION ON ENZYME ACTIVITY
Instructor: MSc. Le Tran Hong Ngoc

Date of submission: June 12th, 2020

Score: ________________

Group 5’s members:

Pham Thi Linh Chi BTBCIU17061

Nguyen Thanh Hang BTBCIU17050

Le Thanh Han BTBCIU17004

Nguyen Thi Ngoc Linh BTBCIU17041

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 International University School of Biotechnology
Vietnam National University – HCMC Department of Biochemistry

I. Abstract
Amylopectin
Enzyme activity can be affected by a variety factor and one of those is enzyme concentration. The purpose
of this lab is to investigate the impact of enzyme concentration. Enzymes can work best within sub-optimal
conditions. So, by determining factors, we can control the enzyme activity. In this experiment, we collected
the absorbance of the enzyme in different concentrations. The enzyme was tested by using 0.001, 0.003,
0.004, 0.005, 0.006 dilution factor. As a result, the enzyme activity is affected by the enzyme concentration.

II. Introduction

The digestion of dietary starch in humans is initiated by salivary enzyme which is called amylase produced
in the salivary glands and the pancreas. Specifically, β-Amylase is glucose-polymer cleavage enzyme that is
responsible for the hydrolysis of α 1-4 glycosidic linkage in the amylose and amilopectin components of
starch. Maltose and dextrin are 2 sugars products of the reaction between β-Amylase and starch. There are
differences between maltose and dextrin: Maltose consists of two glucose units in α -1,4 linkage while dextrin
is made up of several glucose units joined by α -1,6 linkage in addition to α -1,4 linkage.

By far the most common storage of polysaccharide in plants is starch, which exists in two form: α-amylose
and amylopectin. Most forms of starch in nature are 10% to 30% α-amylose and 70% to 90% amylopectin. α-
amylose is composed of linear chains of D-glucose in α -1,4 linkages. Amylopectin is a highly branched chain
of glucose units. The linear linkages in amylopectin are α -1,4, whereas the branch linkages are α -1,6.

Amylose Amylopectin
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 International University School of Biotechnology
Vietnam National University – HCMC Department of Biochemistry

There are several factors that affect the enzymatic reaction rate such as enzyme concentration, substrate
concentration, temperature, pH, and heavy metal cations. The experiment of this lab work focus on how
enzyme concentration would affect the reaction rate.

III. Materials and methods

1. Materials and equipment

The chemical used in the experiment were saliva of random member in group, substrate (starch), activator
(sodium chloride), inactivator (sodium hydroxide), DNS, and distilled water. The equipment used in the
experiment were test tube, beaker, pipette P1000, pipette P100, glass pipette 5 mL, 1 mL; and the UV-Vis
machine.

2. Methods

Firstly, 1 mL of saliva was taken into a small beaker and diluted to 20 mL (added 19 mL of H2O). Then, 11
test tubes were set up. There were 1 tube marked for overall blank, 5 tubesmarked for blank, and 5 tubes
marked for sample. 2.5 mL buffer were added in all test tubes. Next, 2.5 mL substrate were added in all the
tubes, except the overall blank. Sodium chloride as an activator were filled to all tubes by 1 mL. Before going
to the incubation process at 37oC for 15 minutes, each sample tube was added by enzyme with different
volume. The enzyme volume put into sample tube 1st, 2nd, 3rd, 4th, 5th was 0.2 mL, 0.4 mL, 0.6mL, 0.8 mL
and 1 mL, respectively.

After incubation process completed, apart from 5th blank and sample, distilled water were added to each
tubes with the following volume showed in the table below:

Overall Blank Sample Blank Sample Blank Sample Blank Sample

blank 1 1 2 2 3 3 4 4

Volume of
distilled 1.0 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2
water (mL)

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 International University School of Biotechnology
Vietnam National University – HCMC Department of Biochemistry

Then, 0.5 mL of sodium hydroxide as inactivator was added to each tube. The enzyme solution was added
into each blank with different volume. Blank tube 1st, 2nd, 3rd, 4th, 5th was 0.2 mL, 0.4 mL, 0.6mL, 0.8 mL
and 1 mL of enzyme, respectively. A detector DNS was added about 0.5 mL to each test tube. All the tubes
were going to boiling process for 5 minutes at 95oC.
All the tubes were recorded the absorbance light at 540 nm so that it could point out the different between
optimal density of each tube

III. Results

Results after running the blank in spectrophotometer

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 International University School of Biotechnology
Vietnam National University – HCMC Department of Biochemistry

Results after running the sample in spectrophotometer

Table 1. Data sheet of optical density at 540 nm

Tube
Blank 1 Smaple 1 Blank 2 Sample 2 Blank 3 Sample 3 Blank 4 Sample 4 Blank 5 Sample 5
Time
1. 0.406 0.701 0.392 0.699 0.404 0.725 0.419 0.708 0.413 0.697

2. 0.405 0.701 0.391 0.698 0.403 0.724 0.418 0.708 0.415 0.696

3. 0.405 0.702 0.392 0.698 0.404 0.725 0.419 0.708 0.413 0.696

Mean 0.405 0.701 0.392 0.698 0.404 0.725 0.419 0.708 0.414 0.696

Δ OD 0.296 0.306 0.321 0.289 0.282

0.001 0.003 0.004 0.005 0.006

Dilution factor

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 International University School of Biotechnology
Vietnam National University – HCMC Department of Biochemistry

݁ ݊ ‫݁ݖ‬ ݁ ݊ ‫݁ݖ‬
Dilution factor = x
݉ ݉ f
= x

Δ OD = OD sample – OD blank

Graph 1: Dilution factor vs absorbance

Optimum dilution factor for enzyme salivary amylase is 0.004 (4:1000) because the absorbance value of
sample 3 is the highest.
IV. Discussion
1. Evaluate the assay
In the experiment, all test tubes are incubated at 37 0C for 15 minutes because the optimal temperature from
370C to 42 0C made the activity of amylase is highest.
DNS reagent plays a role in stopping the reaction occurs, by that way, the spectrophotometer can allow the
light of the formed glucose to be read. 3-amino-5-nitrosalicylic acid, which absorbs light at 540 nm is formed
when DNS reacts with reducing sugars (glucose). By heating for 5 minutes at elevated temperature (100 0C)
the alkaline mixture of glucose with DNS (yellow color), the red-brown color developed will absorb the light
mostly.

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 International University School of Biotechnology
Vietnam National University – HCMC Department of Biochemistry

According to graph 1, the dilution factor from 0.003 to 0.004 would be ideal, as it gives a large optimal
density (0.306 - 0.321). Dilution factors in the range between 0.004 and 0.007 (i.e. the region with reduced
slope) will underestimate the true level of enzyme activity because some problems may happen during the
procedure that affected the results.
2. Possible errors
2.1 - Personal error
The lab work required in pipette-technique skill in order to minimize the errors. If pumping pipette strongly,
the enzymes in solution could be easily broken. Another reason is that the extreme solution has a very low
concentration of enzyme, so the enzyme can be broken down too.
2.2 - Random error
Enzymes can denature over time, especially when they are too diluted and the products of some reactions can
inhibit the enzyme.
V. Conclusion
Overall, the objective of the lab was met in determining the effects of enzyme concentration on the reaction
rate. All this data that was collected was used to agree on the objectives of the experiment but not showing a
correct trend in data. To make these results correct with the hypothesis, it would be necessary to complete
the experiment multiple more times because some sections cannot verify the results completely.
VI. References:

1. Kaplan, F., & Guy, C. L. (2004). β-Amylase induction and the protective role of maltose during
temperature shock. Plant physiology, 135(3), 1674-1684.
2. Whistler, R. L., & Daniel, J. R. (2000). Starch. Kirk‐othmer encyclopedia of chemical technology.