Amylase is an enzyme that catalyzes the hydrolysis of starch into sugars. Amylase is present in the saliva of humans and some other mammals, where it begins the chemical process of digestion. Foods that contain large amount of starch but less amount of sugar, such as rice and potatoes, may acquire a slightly sweet taste as they are chewed because amylase degrades some of their starch into sugar. α- amylase is a protein enzyme that hydrolyses alpha bonds of large, alpha - linked polysaccharides, such as starch and glycogen, yielding glucose and maltose. It is a major form of amylase found in humans and other mammals. Many of the enzymes used in the industries are extracellular derived from microorganisms. Among various extracellular enzymes, alpha amylase ranks first in terms of commercial exploitation. Bacteria and fungi secrets amylases to the outside of the cells to carryout extracellular digestions when they have broken down the soluble starch, the soluble end products such as Glucose or Maltose are absorbed into their cells. The industrially important Bacillus strains which are extensively used to produce alpha amylase, are, B. licheniformis, B. subtilis etc. B. amyloliquefaciens Bacillus licheniformis is a Gram-positive endospore forming organism that can be isolated from soils and plant material all over the world. This organism is used extensively for large-scale industrial production of exoenzymes as it can secrete large quantities of proteins of up to 20–25 g/l. The use of the submerged culture is advantageous because of the ease of sterilization and its process control. The objective of this work was to study the pattern and the comparison of α-amylase production by using two strains of Bacillus licheniformis, MTCC 2617 and MTCC 2618 using four different substrates starch, rice, wheat and ragi powder as carbon source. Ragi or finger millet is round, soft yet firm and rich brown in color. It is probably the only edible solid you are advised to swallow not chew. A gram of ragi has 72% carbohydrate, 3.6% fiber, 7.3% of protein, vitamin B and a good combination of minerals.

1. COMPARATIVE STUDY ON
PRODUCTION OF α-AMYLASE
FROM BACILLUS
LICHENIFORMIS STRAINS:
By Priya Paul, Asmita Raha, Sudipta Das, Avirup Dutta.

2. Priya Paul
M.Sc Biotechnology 1st Semester
2
1

3. Index:
▰ Introduction.
▰ Materials and methods.
▻ Microorganisms and Maintenance of Culture.
▻ Inoculum and Production Media.
▻ Extraction of the α-Amylase Enzyme.
▰ RESULTS AND DISCUSSION.
▻ Starch Hydrolysis Test.
▻ Enzyme Production.
▰ Characterization of enzyme.
▻ Effect of pH and Temperature on Enzyme Activity.
▻ Effect of Activator and Inhibitor on Enzyme Activity.
▻ Effect of Substrate Concentration on Enzyme Activity. 3

4. INTRODUCTION:
▰ Amylase is an enzyme that catalyzes the hydrolysis of starch into
sugars.
▰ Amylase is present in the saliva of humans and some other
mammals, where it begins the chemical process of digestion.
Foods that contain large amount of starch but less amount of
sugar, such as rice and potatoes, may acquire a slightly sweet
taste as they are chewed because amylase degrades some of
their starch into sugar.
▰ α- amylase is a protein enzyme that hydrolyses alpha bonds of
large, alpha - linked polysaccharides, such as starch and
glycogen, yielding glucose and maltose. It is a major form of
amylase found in humans and other mammals.
4

5. Contd:
▰ Many of the enzymes used in the industries are extracellular derived from
microorganisms. Among various extracellular enzymes, alpha amylase ranks
first in terms of commercial exploitation.
▰ Bacteria and fungi secrets amylases to the outside of the cells to carryout
extracellular digestions when they have broken down the soluble starch, the
soluble end products such as Glucose or Maltose are absorbed into their
cells.
▰ The industrially important Bacillus strains which are extensively used to
produce alpha amylase, are, B. licheniformis, B. subtilis etc. B.
amyloliquefaciens
5

6. Contd:
▰ Bacillus licheniformis is a Gram-positive endospore forming
organism that can be isolated from soils and plant material
all over the world.
▰ This organism is used extensively for large-scale industrial
production of exoenzymes as it can secrete large quantities
of proteins of up to 20–25 g/l.
▰ The use of the submerged culture is advantageous because
of the ease of sterilization and its process control.
6

7. Contd:
▰ The objective of this work was to study the pattern and the comparison of α-amylase
production by using two strains of Bacillus licheniformis, MTCC 2617 and MTCC 2618
using four different substrates starch, rice, wheat and ragi powder as carbon source.
▰ Ragi or finger millet is round, soft yet firm and rich brown in color. It is probably the only
edible solid you are advised to swallow not chew. A gram of ragi has 72% carbohydrate,
3.6% fiber, 7.3% of protein, vitamin B and a good combination of minerals.
7

8. Asmita Raha
M.Sc Biotechnology 1st Semester
8
2

9. MATERIALS AND METHODS:
▰ Microorganisms and Maintenance of Culture:
Bacillus licheniformis strain MTCC 2617 and MTCC 2618 were procured from Microbial Type
Culture Collection (MTCC) Chandigarh and grown on nutrient agar slants at 37°C and
subcultured every month.
▰ Inoculum and Production Media:
▻ The inoculum was prepared by the addition of sterile distilled water in to the freshly
grown agar slants, from this 0.5ml of suspension was inoculated in to 100ml of sterilized
fermentation medium and incubated in a shaking incubator at 100 rpm at 37oC.
9

10. Contd:
▻ The composition of media was (g/l)
For the production of the enzyme, broths were prepared using four different
substrates Starch, Rice, Wheat and Ragi powder.
▻ The activity of the enzyme produced in the media was checked using enzymatic
assay method at regular time intervals of 24h, 48h, 72h, 96h and 120h
respectively, to find the time period and the medium with the substrate that
showed the highest enzyme production.
10

11. Contd:
11
▰ Extraction of the α-Amylase Enzyme:
▻ After incubation, the culture broth was centrifuged at 5000 rpm for 20 minutes at 4°C.
▻ The supernatant was collected and the amylase activity was estimated .
▻ The supernatant was further purified to obtain the crude enzyme.
▻ The crude enzyme was further purified by ammonium sulfate precipitation, dialysis and ion-
exchange chromatography to obtain pure enzyme.

12. RESULTS AND DISCUSSION:
▰ Starch Hydrolysis Test:
▻ The ability to degrade starch is used as a criterion for the determination of amylase production by a
microbe.
▻ In the laboratory setting, it is tested by performing starch hydrolysis test or starch test to determine the
presence or absence of amylase enzyme.
▻ The test utilizes iodine as an indicator. Starch in the presence of iodine produces dark blue colouration
of the medium as iodine is trapped in the helical structure of starch and a yellow zone or clear zone around a
colony in a blue medium indicates amylolytic activity.
▻ The clear zone indicates hydrolysis of starch into monosaccharides which cannot bind the iodine
molecule and appear as clear zone around the bacterial growth.
▻ Size of the clear zone is directly proportional to the starch hydrolyzing activity of the strain under study.
12

13. Contd:
▻ The strains showed formation of a halo zone, which
indicated the degradation of the starch from the starch
agar by the enzyme α-amylase produced by Bacillus
licheniformis.
13

14. Sudipta Das
M.Sc Biotechnology 1st Semester
14
3

15. Contd:
▰ Enzyme Production:
o In submerged fermentation, the production of the α- amylase by the B.licheniformis MTCC strain 2617, using four different
substrates as carbon source, reached maximum on the third day.
o The production declined by reaching the minimum on the fifth day. Of the four substrates used, starch gave the highest
production of the enzyme on all the days .
15

16. Contd:
16

17. Contd:
o Similarly by submerged fermentation the production of α- amylase by the B.licheniformis MTCC strain 2618,
using four different substrates as carbon source, reached maximum on the 3rd day.
o The production declined by reaching the minimum on the 5th day. The production of the α-amylase from the two
B.licheniformis strains used, MTCC strain 2618 showed the highest production on all the four substrates used as
carbon source, i.e., on the 3rd day, the maximum production of the enzyme was seen and of the substrates used the
medium containing starch gave the highest production of enzyme.
17

18. Contd:
18

19. Characterization:
▰ Effect of pH on Enzyme Activity:
▻ Enzymes are affected by changes in pH.
▻ The most favorable pH value - the point where the enzyme is most active - is known as the optimum pH.
▻ Extremely high or low pH values generally result in complete loss of activity for most enzymes. pH is also a factor in
the stability of enzymes. As with activity, for each enzyme there is also a region of pH optimal stability.
▻ The activity of the enzyme gradually increased, reaching the maximum at pH 7 (As shown in the figure). Further
increase in the pH resulted in the decrease in the activity of α-amylase . The enzyme activity was found to be the maximum
at pH 7 as reported by Vidyalakshmi et al. 2009.
19

20. Contd:
20

21. Avirup Dutta
M.Sc Biotechnology 1st Semester
21
4

22. Contd:
▰ Effect of Temperature on Enzyme Activity:
▻ Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the
temperature is raised. A ten degree Centigrade rise in temperature will increase the activity of
most enzymes by 50 to 100%.
▻ Variations in reaction temperature as small as 1 or 2 degrees may introduce changes of 10
to 20% in the results.
▻ In the case of enzymatic reactions, this is complicated by the fact that many enzymes are
adversely affected by high temperatures. As shown in Figure , the reaction rate increases with
temperature to a maximum level, then abruptly declines with further increase of temperature.
Because most animal enzymes rapidly become denatured at temperatures above 40°C.
▻ Over a period of time, enzymes will be deactivated at even moderate temperatures. Storage
of enzymes at 5°C or below is generally the most suitable. Some enzymes lose their activity when
frozen.
▻ The bacterial amylases have an optimum temperature in range of 30-100°C .
22

23. Contd:
23

24. Contd:
▰ Effect of activator on enzyme activity:
The activity of α-amylase decreased with the increase in concentration of the inhibitor.
24

25. Contd:
▰ Effect of inhibitor on enzyme activity:
The activity of α-amylase decreased with the increase in concentration of the inhibitor.
25

26. ▰ Effect of substrate concentration on enzyme activity:
The activity of α-amylase showed increased with increase in the substrate concentration.
26
Contd:

27. 27