Alpha-Amylase Production

by Bacillus licheniformis
1331using Jackfruit Seeds
as Substrate

Rica Mae A. Palongpalong

Prof. Percival G. Garcia
What are Amylases?
•Enzymes that breakdown starch
glycogen
•These are produced by living
organisms such as bacteria,
plants, and humans.
•Bacteria and fungi secrete
amylases to the outside of the
cells to carry out extracellular
digestion.
•When they have broken down
the insoluble starch, the soluble
end products such as glucose or
maltose are absorbed into their
cells.
 Classification of Amylases
(based on how organisms breakdown starch
molecules)
1. Alpha-amylase- reduces the viscosity of starch by
breaking down the bonds at random, therefore
producing varied sized chains of glucose
2. Beta-amylase- breaks the glucose-glucose bonds
by removing two glucose units at a time, thereby
producing maltose
3. Amyloglucosidase- breaks successive bonds from
the non-reducing end of the straight chain,
producing glucose
Why bother about Amylases?
1. Additives to detergents
2. Textile desizing
3. Paper industries
4. Starch liquefaction/
saccharification
5. Food, adhesive, sugar production
6. Brewing and distilling industries
7. High fructose corn syrup preparation
8. Clinical, medicinal, and analytical
chemistry
 Jackfruit
•Scientific name: Artocarpus
heterophyllus
•Local name: Nangka or langka
•Widely grown in South and Southeast
Asia including Philippines, and it is the
national fruit of Bangladesh and
Indonesia
•Ripe fruit contains well flavored
yellow sweet edible bulbs with seeds
inside it. Seeds are normally
discarded, some are boiled or roasted
for snacks.
•In Thailand seeds are made into flour.
Jackfruits food composition per 100 gram edible portion
consists of the following:
FRUIT
CONSTITUENT IMMATURE RIPE (PULP) SEE
Moisture (%) 85.2 72.4
Food Energy, cal. 51 97
Protein, g 2 1.4
Fat, g 0.6 0.4
Total carbohydrates, g 11.5 24
Fiber, g 2.6 0.8
Ash, g 0.7 1
Calcium, mg 53 23
Iron, mg 0.4 1.1
Sodium, mg 3 2
Potassium, mg 323 107
Vit. A I.U. 30 175
Thiamine, mg 0.12 0.9
Rivoflavin, mg 0.05 0.05
Bacillus licheniformis
• Gram-positive thermophilic bacterium
•Motile
•Spore-forming
•Facultatively anaerobic-rod
•Commonly found in the soil and bird
feathers
•Optimal temperature for enzyme
secretion: 37°C
•Optimal growth temperature is around
50°C
•Show high enzyme capacity
production during exponential growth
phase
 Objectives
•To determine alpha-amylase production by
Bacillus licheniformis using an experimental
substrate
•To know whether jackfruit seeds exhibit the
essential characteristics of a good solid substrate
for alpha-amylase production
 Scope and Limitations
This study will include only:
1. Production of crude amylase
2. Optimization of cultural and environmental
parameters such as pH, temperature,
carbon/nitrogen ratio
This study will not include:
1. Purification of crude amylase
 Significance of the Study
•Contribution to industrial biotechnology- The result of
this exploratory study may lead to a discovery of a
potential substrate for enzyme production for
commercial use.
•It will contribute to waste management program since
jackfruit seeds are normally discarded as waste product.
•It has economic value to fruit vendors and farmers. If
proven to be good substrate source then it will become
a potential source of income.
 Preparation of jackfruit seed samples

Bacteria strain

Preparation of nutrient agar and nutrient broth

Preparation of inoculum
 Production medium

Jackfruit seeds Yeast extract powder pH Temperature

10% 15% 20% 20% 7 30C 37C

The experiment was done in triplicate analysis.

 Preparation of crude enzyme extract

Bradford assay Alpha-amylase assay Biomass

Iodine reagent Buffer solution Starch solution

Analytical procedure
 Results for Alpha-amylase assay at pH 7 and 30C
A b s o r b a nAc l ep h a - a m y l a s eA
S u b s t r a t e C 0o .n0 t1r 5o 7l
1 0 % C a r b o n , 2T 0r i%a l N1 i t r o g e n0 . 0 1 7 4 -0 .1 0 8 2 8 0 5
T ria l 2 0 .0 1 2 6 0 .1 9 7 4 5 2 2 2
T ria l 3 0 .0 1 3 8 0 .1 2 1 0 1 9 1 0

1 5 % C a r b o n , 2T 0r i%a l N1 i t r o g e n0 . 0 1 4 7 0 .0 6 3 6 9 4 2 6
T ria l 2 0 .0 1 4 5 0 .0 7 6 4 3 3 1 2
T ria l 3 0 .0 1 5 1 0 .0 3 8 2 1 6 5 6

2 0 % C r a b o n , 2T 0r i%a l N1 i t r o g e n0 . 0 1 3 9 0 .1 1 4 6 4 9 6 8
T ria l 2 0 .0 1 4 7 0 .0 6 3 6 9 4 2 6
 Results for Alpha-amylase assay at pH 7 and 37C
A b s o r b a nAc l ep h a - a m y l a s eA
S u b s t r a t e C 0o .n0 t4r 3o 4l
1 0 % C a r b o n , 2T 0r i%a l N1 i t r o g e n0 . 0 1 8 3 0 .5 7 8 3 1 0 1 3
T ria l 2 0 .0 1 5 5 0 .6 4 2 8 5 7 1 4
T ria l 3 0 .0 1 3 4 0 .6 9 1 2 4 4 2 3

1 5 % C a r b o n , 2T 0r i%a l N1 i t r o g e n0 . 0 2 4 3 0 .4 4 0 0 9 2 1 6
T ria l 2 0 .0 1 5 1 0 .6 5 2 0 7 3 7 3
T ria l 3 0 .0 1 1 5 0 .7 3 5 0 2 3 0 4

2 0 % C r a b o n , 2T 0r i%a l N1 i t r o g e n0 . 0 4 0 1 0 .0 7 6 0 3 6 8 6
T ria l 2 0 .0 3 1 2 0 .2 8 1 1 0 5 9 9
 Results for Bradford assay at pH 7 and 30C

a b s o r b acno cnec e n t r a t i oA nv
1 0 % C a r b o n , 2 0 %T r Ni a il t r1 o g e n1 . 2 4 5 9 3 0 8 4
T ria l 2 1 .3 0 8 3 2 3 9 .2 5 3 1
T ria l 3 1 .2 4 3 6 3 0 7 8 .2 5

1 5 % C a r b o n , 2 0 %T r Ni a il t r1 o g e n1 . 3 3 8 7 3 3 1 6
T ria l 2 1 .2 4 0 7 3 0 7 1 33
T ria l 3 1 .4 3 7 6 3 5 6 3 .2 5

2 0 % C a r b o n , 2 0 %T r Ni a il t r1 o g e n1 . 2 6 8 7 3141
T ria l 2 1 .2 7 6 7 3161 31
 Results for Bradford assay at pH 7 and 37C

a b s o r b acno cnec e n t r a t i oA nv
1 0 % C a r b o n , 2 0 %T r Ni a il t r1 o g e n1 . 1 6 6 9 2 8 8 6 . 5
T ria l 2 1 .2 1 7 5 3 0 1 3 28
T ria l 3 1 .1 1 2 5 2 7 5 0 .5

1 5 % C a r b o n , 2 0 %T r Ni a il t r1 o g e n1 . 0 2 0 5 2 5 2 0 . 5
T ria l 2 1 .1 0 3 9 2 7 2 9 2
T ria l 3 1 .2 6 2 3 3 1 2 5

2 0 % C a r b o n , 2 0 %T r Ni a il t r1 o g e n1 . 3 2 2 5 3 2 7 5 . 5
T ria l 2 1 .3 1 1 4 3 2 4 7 .7 5 3 0
 Results for Alpha-amylase assay, Bradford assay, and Biomass at regular
intervals

T i m e A ( lhp ) h a - a m y l a s e Pa rcot it ve i tny C( Uo /nmc le) n Bt r iaot mi o

0 0 .2 8 7 9 2 .6 9 8 8 0.
1 0 .3 1 6 5 2 .9 7 4 5 0 .2
2 0 .3 2 7 5 3 .0 4 5 5 0.
4 0 .4 0 4 5 3 .2 8 8 8 0 .3
6 0 .5 8 6 8 3 .3 2 8 8 0 .3
8 0 .6 4 8 4 3 .2 2 4 0.
18 0 .6 5 9 3 3 .2 8 2 5 0 .2
24 0 .6 5 9 3 3 .2 9 7 5 0 .3
27 0 .6 6 5 9 3 .2 3 1 8 0 .1
 3 .5
3 A lp h a -a m y la s
a c tivity (U /m l)
2 .5
Concentrations (mg/ml)

2 P ro te in C o n c e
(m g /m l)
1 .5
1 B io m a s s
C o n c e n tra tio n
0 .5 (m g /m l)
0
0 10 20 30 40
tim e (h )
 Conclusion
1. Jackfruit seed can be a carbon source in alpha-
amylase production.
2. Alpha-amylase activity was optimum at 10% carbon,
20% nitrogen, pH 7, and 37C.
3. Alpha-amylase activity was highest at 4-8 hours of
fermentation.
 Recommendation
• Try other factorial design and other less expensive
nitrogen sources such as corn steep liquor and
peptone.
• It is also recommended that other investigators on
the purification of alpha-amylase, characterization of
alpha-amylase by using SDS-PAGE, and the
process of its production in commercial volume shall
conduct a follow up study.
Thank you