INTRODUCTION

As diastase, amylase was the first enzyme to be discovered and isolated (by Anselme Payen
in 1833). All amylases are glycoside hydrolases and act on α-1,4-glycosidic bonds. Amylases
are significant enzymes for their specific use in the industrial starch conversion process.
Amylolytic enzymes act on starch and related oligo- and polysaccharides. In the food
industry amylolytic enzymes have a large scale of applications, such as the production of
glucose syrups, high fructose corn syrups, maltose syrup, reduction of viscosity of sugar
syrups, reduction of turbidity to produce clarified fruit juice for longer shelf-life,
solubilisation and saccharification of starch in the brewing industry. The baking industry uses
amylases to delay the staling of bread and other baked products; the paper industry uses
amylases for the reduction of starch viscosity to achieve the appropriate coating of paper.
Amylase enzyme is used in the textile industry for warp sizing of textile fibers, and used as a
digestive aid in the pharmaceutical industry (Akansha, & Varsha, 2013).

DISCUSSION:

Amylase of fungal origin was found to be more stable than the bacterial enzymes on a
commercial scale; many attempts have been made to optimize culture conditions and suitable
strains of fungi. Moulds are capable of producing high amounts of amylase; Aspergillus niger
is used for commercial production of α-amylase. Production of fungal amylases especially in
laboratory scale is mainly on Aspergillus niger, probably because of their ubiquitous nature
and non-fastidious nutritional requirements of these organisms. It is possible to enlist the use
of amylases under extreme condition of pH and temperature using thermo-acidophilic and
alkaline amylases. Since the most effective preparation of some applications contain other
enzymes, especially amyloglucosidases and submerged methods give a narrow spectrum of
additional enzymes and it is worthwhile to isolate suitable strains of Aspergillus niger for
efficient mechanism (Suganthi et. al, 2011). The production of α-amylase by moulds has
been greatly affected by cultural and nutritional requirement. This can be seen in figure 1.1,
where the fermented Aspergillus niger into two different shake flask shows different colour
supernatant although the strain was isolated from same plate, the difference might because of
the nutritional value or temperature or pH in both shake flask. That’s why the both
supernatant was tested simultaneously for the presence of amylase enzyme in separate tubes,
but there was no different in results. The colour difference might just indicate the
concentration of the enzyme released by the strains.
The presence or absence of starch in the solutions was tested using iodine. Iodine forms a
dark blue or purple complex with starch, but does not react with diastases. If iodine is added
to a diastases solution, the only colour seen is light blue or sometimes colourless. Therefore,
the faster the dark blue colour of starch is lost, the faster the enzyme amylase (diastases) is
working. If the amylase is inactivated, it can no longer hydrolyse starch, so the blue colour of
the starch-iodine complex will persist. The iodine (I3- and I5- ions) fit inside the coils of
amylose, the charge transfers between the iodine and the starch, and the energy level spacing
in the resulting complex correspond to the absorption spectrum in the visible light region
(Somogyi, 1938).

During catalysis, the first step is the substrate (S) binding to the enzyme (E), giving an
enzyme-substrate complex (ES). This is an equilibrium reaction, and will be favoured by a
high concentration of enzyme and/or substrate. After the substrate is bound, the reaction takes
place, and then the product is released (Akansha & Varsha, 2013). According to the result
obtained (refer figure 1.3), the colour change was not so clear especially on the test tube I for
both sample (A&B) which should show light blue colour prior to presence of diastase enzyme
while the colour produced was not so light but it has distinct between test tubes II and III.
This happens because; the volume of supernatant or enzyme used was less to show the
reaction clearly. The doubtful result can be confirmed by adding some more supernatant to
see the reaction and colour change more clearly and there was a presence of diastases in both
samples in test tube I. Besides that, the low colour intensity also might happen due to short
incubation period after added substrate (starch) into test tubes. This is because, the time (5
minutes) framed for the incubation after adding substrate is to allow the enzyme in the tubes
to binds to the substrate and breakdown it to product (maltose).

All reactions are faster at a higher temperature. However, enzyme-catalysed reactions

become slower or stop if the temperature becomes too high, because enzymes become
denatured at high temperatures. Therefore, enzymes have an optimum temperature that
corresponds to maximum activity. The optimum temperature is usually around body
temperature (37°C) (Suganthi et. al, 2011). Based on the theory, we know that the enzyme
cannot function or denatured at high temperature. So to valid our result or to obtain only
enzyme as a product, we confirmed with test tubes II. If starch is subjected to dry heat, it also
can be break down to form dextrin which will further broken down into maltose that can
create false positive result. To ovoid this from occur, the enzyme added test tubes II will
heated until boil to denature the enzymes, before addition of starch. Then, the starch is added
and test with Iodine, if it shows light blue colour as the test tubes I, it means the starch was
already broken down into maltose. So, we should repeat the experiment by using new starch
solution, to further confirm that the starch is the cause of the false positive. According to our
result (figure 1.3), the test tubes II shows dark blue colour and also some suspension particles
floating on it which means the enzymes was denatured completely and it did not broke any
starch.

Conclusion:

The present study showed that the isolated Aspergillus niger able to produced appreciable
amylase (diastases) enzyme but not in large amount. Further, the study also revealed that
amylase enzyme produced by the isolated fungi confirmed by the colour change using starch-
iodine test after enzyme-substrate catalyst.

REFERNCES:

Akansha, K., & Varsha, N. (2013). Production of Amylase Enzyme by Isolated

Microorganisms and It’s Application. International Journal of Pharmacy and
Biological Sciences, vol. 3 (4), pp. 354-360. Retrieved from
https://www.ijpbs.com/ijpbsadmin/upload/ijpbs_5387f2cb9c9a2.pdf
Somogyi, M. (1938). Analysis of Diastatic Split-Products of Starch. Journal of Biology
Chemistry, vol. 124, pp. 179-187. Retrieved from
http://www.jbc.org/content/124/1/179.full.pdf
Suganthi, R., Benazir, J. F., Santhi, R., Ramesh, V., Anjana, H, Nitya, M., Nidhiya, K. A.,
Kavitha, G., & Lakshmi, R. (2011). Amylase Production by Aspergillus niger Under
Solid State Fermentation Using Agroindustrial Wastes. International Journal of
Engineering Science and Technology, vol. 3 (2), pp. 1756-1763. Retrieved from
https://www.researchgate.net/profile/
RESULTS:

Sample A

Sample B

Figure 1.1: Shows fermented Aspergillus niger into two different shake flask with different
colour of supernatant.

1A 1B 2A 2B 3

Figure 1.2: Shows comparison of colour change before and after iodine added for each test