VIETNAM NATIONAL UNIVERSITY – HO CHI MINH CITY

INTERNATIONAL UNIVERSITY

Experiment 2
BIOSYNTHESIS OF ENZYMES FROM FUNGI
AND BACTERIA
AND ENZYME ACTIVITY DETERMINATION

Enzymology – Spring 2020

Lab session – Friday morning

Group member: Phan Hoang Thien An – BTBCIU17022

Lam Ngoc Ngan Anh – BTBCIU17014
Phan Viet Ha – BTBCIU15052
Nguyen Hoang Long – BTBCIU15025
Nguyen Huynh Thanh Thao – BTBCIU16084

Supervisor: MSc. Le Tran Hong Ngoc

Teaching assistant: Truong Phu Le

June 5th, 2020

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 TABLE OF CONTENTS

ABSTRACT

1. Introduction ....................................................................................... 4

2. Materials and methods ......................................................................... 5

Standard curve construction .......................................................... 5

Enzyme extraction and measurement preparation ............................ 5

Calculation of enzyme activity ........................................................ 5

3. Results .............................................................................................. 6

Glucose standard curve ................................................................. 6

Enzyme activity estimation ............................................................ 6

4. Discussion .......................................................................................... 7

5. Conclusion ......................................................................................... 8

REFERENCES

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Experiment 2
BIOSYNTHESIS OF ENZYMES FROM FUNGI AND BACTERIA
AND ENZYME ACTIVITY DETERMINATION

ABSTRACT

In this experiment, amylases – enzymes that break down starch molecules, were
produced from bacteria and fungi feeding on rotten potato. To estimate the
enzyme activity, the sample was compared with a blank in which the enzyme had
been inactivated. DNS reagent was utilized to stop the starch hydrolysis and reflect
the glucose amount produced in terms of color which was determined at 540nm.
To convert color intensity to reducing sugar equivalent, a concentration glucose
calibration curve was constructed. The enzyme activity was defined as the amount
of glucose produced in the presence of enzyme in a specific period of time.
According to the obtained result, the real absorbance value of sample was negative
and there was no difference in color between blank and sample tubes. Therefore,
the amount of glucose and the enzyme activity were unidentified.

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 1. Introduction
Amylases are a group of enzymes that hydrolyse starch into smaller
carbohydrate molecules such as maltose or glucose. Amylases are divided into
three groups, namely -amylase produced by salivary glands and pancreas that
breaks down starch randomly into different sized chains, -amylases present in
yeasts, moulds, bacteria, and plants, particularly in the seeds that creates mostly
maltose and amyloglucosidases can cleave non-reducing end bonds, which
produce glucose monomers.

Figure 1. General illustration of starch hydrolysis by -, -amylase, and

amyloglucosidase.

Enzymes are generally extracted from cells of microorganisms by the

autolysis of the cell walls or by using a lysis-promoting-agent such as sodium
dodecyl sulphate, and then removed from other cell debris. Other proposed and
utilized methods are mechanical destruction of the cell walls by trituration,
ultrasonication or the freezing and thawing method, as well as the employment of
lysozyme to destroy the cell walls enzymatically.
Two major groups of microorganisms which play important roles in amylase
production are bacteria and fungi. They secrete amylases out of their cells to carry
out extra-cellular digestion. Additionally, the level of amylases produced depends
on the microbial origin, where strains isolated from starch or amylose-rich
environments naturally produce higher amounts of this enzyme. Other factors also
play vital roles in the rate of amylase production such as pH, temperature, and
carbon and nitrogen sources [1]
.
Amylase from fungal and bacterial sources are accounted for approximately
25% of the world enzyme market [2,3]
. Humans have been applying this enzyme
for many purposes such as brewing, high fructose corn syrup preparation,

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additives to detergents for removing stains and saccharification of starch for
alcohol production [4]
.
In this experiment, dinitro salicylic acid (DNS) colorimetric method is used
to determine the amylase activity. In alkaline condition, 3,5-dinitrosalicylic acid
(yellow) is reduced to 3-amino-5-nitrosalicylic acid (reddish brown) while the
carbonyl group on reducing sugars is oxidized to carboxyl group. Then, the color
intensity is measured by UV-Vis spectrophotometer at 540 nm.

2. Materials and methods

Standard curve construction
Glucose standard curve was constructed by adding DNS reagent to glucose
at known concentrations from 0 to 12 mM. Absorbance of glucose standard
solutions were read at 540 nm in a spectrophotometer.

Enzyme extraction and measurement preparation

Amylase-producing bacterial or fungal culture was centrifuged for 20
minutes at 5000 rpm to collect its enzyme extract in the supernatant.
Test tubes were used to contain a sample and a blank. To 1 mL of enzyme
extract, 1 mL of 1% soluble starch in citrate-phosphate buffer (pH 6.5) was added
and the sample mixture was incubated in a 40 oC water bath for 30 minutes. The
blank was prepared by inactivating 1 mL of enzyme extract in a boiling water bath
for 20 minutes. This boiling tube mouth was covered with aluminum foil while the
sample tube used parafilm instead. One milliliter of starch was subsequently added
to the blank test tube. To each test tube, 2 mL of DNS reagent were added and
they were boiled again for 5 minutes while covered in aluminum foil. After that,
both tubes were cooled and 20 mL of distilled water was added prior to absorbance
reading at 540 nm using a spectrophotometer.

Calculation of enzyme activity

To quantify the amount of glucose produced by the enzyme in bacterial
culture extract, a glucose calibration curve was constructed and applied to convert
color (absorbance value) to reducing sugar equivalent. Enzyme activity was
presented as the amount of glucose produced per mL of enzyme amount in 30
minutes.

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 3. Results
Glucose standard curve

Figure 2. Standard curve of glucose in the presence of DNS reagent. Absorbance

was measured at 540 nm.

Enzyme activity estimation

𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑚𝑀) =
0.2028

Blank Sample Blank Sample

Figure 3. “Blank” and “Sample” Figure 4. “Blank” and “Sample”

tubes after DNS addition. tubes after DNS addition and
boiled for 5 minutes.

Table 2. Absorbance of enzyme extract (inactivated and active) in the presence

of starch and DNS reagent and glucose.

Absorbance (540 nm)

Blank (inactivated enzyme) 0.338 0.338 0.339

Sample (active enzyme) 0.289 0.289 0.289

Real absorbance (of glucose produced) -0.049 -0.049 -0.05

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 4. Discussion
Bacteria or fungi do not normally produce amylase in the presence of
nutrients simpler in structure than starch (e.g., glucose), thus researcher must
remove all other nutrients except for starch in order to force them to produce this
enzyme to hydrolyze starch. In order to extract this extracellular enzyme from
bacteria, high speed centrifugation was well-qualified to obtain the crude enzyme
extract without grinding first and other methods which disrupt the cell membrane
are unnecessary [5,6]
. Moreover, a refrigerated centrifuge is required as a cooling
system so that our desired enzyme is reserved.
In the experiment, the blank which containing 1 mL of enzyme extract must
be boiled to inactivate its enzyme before adding starch. Both sample and blank
were mixed with DNS reagent to stop the hydrolysis reaction and provide color
development so as to be measured by a spectrophotometer. Amylase present in
the enzyme extract hydrolyzed starch to glucose which is a reducing sugar that
gave the red brown color appearance when reacted with DNS reagent [7]
. As a
result, the darker the color was, the higher the amount of glucose and the greater
quantity of amylase presented.

Figure 5. Reduction-oxidation reaction between DNS reagent and reducing

sugars.

In theory, the blank should be observed with the yellow color of DNS
reagent and the glucose-containing sample should be red-brown in color.
However, against all expectations, the blank received a red-brown color similar to
that of the sample. Moreover, the absorbance of the blank when read by the
spectrometer was even higher than that of the sample which resulted in the
negative value of glucose quantity and thus the enzyme activity was unidentified.
The red-brown color appearance in the blank reflected the presence of reducing
sugar in the solution. There are several reasons for this abnormal phenomenon.
First of all, the enzyme extract in the blank might not be inactivated yet. In fact,
when the blank was taken out of the 100oC water bath, it was not really hot. The

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blank might not be heated properly, and thus, the enzyme was still activated and
then hydrolyzed the starch into glucose, which contributed to the red-brown color.
Another reason could be that our blank contained the part of starch which had
quite a lot of reducing ends more than the part in the sample tube. In fact, starch
structure still consists of reducing ends which will give red-brown color when
reacting with DNS reagent [8]
.

Therefore, it is proposed that citrate-phosphate buffer should be used in

the blank instead of starch. The absorbance of starch should be measured
separately and then be subtracted from the sample absorbance.
Furthermore, laboratory conditions did not provide optimal environment for
the bacteria to grow and produce high amount of amylase, which causes the
negligible enzyme activity level. In addition, it is impossible to supply optimal
condition if the amylase-producing bacteria identity is unknown.
The insignificance of color difference might be resulted from improper
micropipette performance such as forgetting to wet the tips, using second stop
instead of first stop, and unequal distilled water added also matters.

5. Conclusion
The enzyme activity of amylases extracted from rotten potato was tested
by comparing the color and measuring the absorbance at 540nm of the blank and
experiment tube. Although the extracted enzyme in the blank was inactivated by
boiling, the color of the mixture in blank and experiment tube was not different.
Besides, the real value of sample was negative. As a result, the amount of glucose
and the enzyme activity were unidentified. The incorrect results might be come
from incompletely inactivated enzyme or starch solution contained high amount

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of part that possessed reducing ends. Moreover, the laboratory conditions were
not good to provide the optimal environment for bacteria to grow and produce the
large amount of amylases with high enzyme activity.

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REFERENCES
[1] Tasnia, I. (2016). Isolation of amylase producing bacteria from soil and
identification by 16S rRNA gene sequencing and characterization of amylase.
Department of Mathematics and Natural Sciences, BRAC University.
[2] Bendelow, V. M. (1963). Modified procedure for the determination of diastatic
activity and α‐amylase activity. Journal of the Institute of Brewing, 69(6), 467-
472.
[3] Gopinath, S. C., Anbu, P., Arshad, M. K., Lakshmipriya, T., Voon, C. H.,
Hashim, U., & Chinni, S. V. (2017). Biotechnological Processes in Microbial
Amylase Production. BioMed research international.
[4] Biosynthesis of enzymes from fungi and bacteria. Enzymology Lab Manual.
International University HCMC-VNU, 2020.
[5] Chisti, Yusuf., & Moo-Young, Murray. (1986). Disruption of microbial cells for
intracellular products . Enzyme Microb. Technol., 8. Retrieved from
https://www.massey.ac.nz/~ychisti/CellDRev.pdf
[6] Luang-In, V., Yotchaisarn, M., Saengha, W., Udomwong, P., Deeseenthum, S.,
& Maneewan, K. (2019). Isolation and Identification of Amylase-producing
Bacteria from Soil in Nasinuan Community Forest, Maha Sarakham, Thailand.
Biomedical & Pharmacology Journal, 12(3), 1061–1068. doi: 10.13005/bpj/1735
[7] Pandey, A. (2010). Enzyme technology. New Delhi: Asiatech Publishers.
[8] Lim Chai Teo M-L and Small D. M. (2012). The effect of granule size on the
digestibility of wheat starch using an in vitro model. World Academy of Science,
Engineering and Technology, 69, 789-793.