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Alpha-amylase Production by Bacillus licheniformis 1331 using

Jackfruit Seeds as Substrate
Rica Mae A. Palongpalong1, Percival G. Garcia2
1
Student, School of Chemical Engineering and Chemistry, Mapua Institute of Technology
2
Adviser, School of Chemical Engineering and Chemistry, Mapua Institute of Technology

Abstract

Amylases have great significance in biotechnology and are commercially important enzymes in
various starch processing industries. The microbial amylases meet industrial demands and have almost
completely replaced chemical hydrolysis of starch in starch processing industry. This study explored the
use of jackfruit seed as carbon source in the production of alpha-amylase. A production medium composed
of ground jackfruit seed and yeast extract powder was mixed with 10% inoculum of Bacillus licheniformis
1331 cultured in nutrient broth at 30°C for 18 hours. The effects of temperature and carbon to nitrogen ratio
were determined. Triplicate analysis was done at 10%, 15%, 20% carbon source with 20% nitrogen source
set at pH 7 and temperature of 30°C and 37°C. It was found out that alpha-amylase production was optimal
at 10% carbon, 20% nitrogen, pH 7, and 37°C. Amylase production was conducted in 1000 ml medium and
incubated with shaking. Samples were taken at regular intervals at 0, 1, 2, 4, 6, 8, 18, 24, 27, and 30 hours
of fermentation. Enzyme activity and protein concentration were determined using Alpha-amylase assay
and Bradford assay, respectively. Biomass was determined by dry cell weight (DCW) analysis. Alpha-
amylase production was highest during exponential growth at 4-8 hours of fermentation.

Keywords: alpha-amylase, Bacillus licheniformis, jackfruit seed, yeast extract

1. Introduction used as a substrate for alpha-amylase
production by Bacillus subtilis under
In the environment abound solid-state fermentation (Krishna and
possible sources of essential materials or Chandrasekaran, M., 1996). A similar
substances necessary for the study was also done using potato
development or production of certain peelings (Shukla, J. and Kar, R., 2000).
products like enzymes, which will be Trypsin/ chymotrypsin inhibitor was
useful to humans, plants, and other isolated from jackfruit by ammonium
animals. These substances can be sulfate fractionation and
produced in significantly large amounts chromatography (Sai Annapurna, S. and
with the advent of biotechnology, but Siva Prasad, D., 1990). These studies
primarily the materials and the factors showed that some enzymes could be
that influence its production source must produced using components of fruits and
be identified. Papain was prepared from vegetables as substrate.
high quality latex of Carica papaya This particular study aims to
(Baines, B. S. and Brocklehurst, K., produce an enzyme by a Bacillus sp.
1978). Some wastes from fruits and using the seeds of a locally grown fruit
vegetables can also be a good substrate as substrate. This particular fruit is
for enzyme production. One study was jackfruit scientifically known as
done on banana fruit stalk, which was Artocarpus heterophyllus or locally
known as “nangka” or “langka”, which No enzyme purification and
is a favorite dessert of Filipinos and is a characterization will be done.
widely grown fruit crop in the The significance of this study is
Philippines. It contains carbohydrates, that another substrate will be added to
protein, calcium, iron, sodium, the existing list of carbon sources for
potassium, B-complex, ascorbic acid, amylase production by microorganisms.
and small amounts of fats, ash, and iron. Many industrial processes use waste
Analysis of jackfruit food composition materials from plants or animals. Part of
per 100 gram edible portion showed that solving the environmental problem of
the carbohydrate content of the seed is waste management can be addressed by
34.90 g (Agriculture and Fisheries the utilization of waste products.
Information Science, Department of Jackfruit seeds as waste from fruit can
Agriculture). be a potential material for enzyme
Based on the above composition, production. Being a substrate it will
the jackfruit seed having a high content become a source of income for fruit
of carbohydrate can be used as a vendors and farmers. This study
substrate for the production of the becomes important to the biotechnology
enzyme amylase. The production of industry and likewise contributes to
amylase is of great significance in environmental management and
biotechnology. It is commercially economic stability.
important in some processing industries
such as beverages, food, textile, 2. Methods
detergents, and paper industries (Pandey
et al., 2000). 2.1 Preparation of jackfruit seed
This enzyme can be produced
using a specific microorganism or fungi One hundred (100) g of jackfruit
under controlled conditions. The seeds was autoclaved in 1L beaker at
amylases can be derived from several 121°C (15 lbs psi) for 15 minutes. The
sources ranging from bacteria to plants outer seed coating was removed. Seeds
to humans. Bacteria and fungi secrete were grinded by using a coffee grinder.
amylases to the outside of their cells to Grinded seeds were placed in zip locked
carry out extracellular digestion. When plastic bag and stored in the freezer.
they have broken down the insoluble
starch, the soluble end products such as 2.2 Bacterial strain
glucose or maltose are absorbed into
their cells. This particular study will use The bacteria, Bacillus
Bacillus licheniformis for amylase licheniformis 1331, was obtained from
production. the Philippine National Collection of
This is an exploratory study to Microorganisms (PNCM), BIOTECH,
determine alpha-amylase production by University of the Philippines, Los
Bacillus licheniformis using jackfruit Baños, Laguna.
seed as substrate. This study is limited to
optimization of cultural and 2.3 Medium Preparation
environmental conditions such as
temperature and carbon/nitrogen ratio. HIMEDIA M001 was used for
maintenance of organism. The
composition of nutrient agar is shown in and HIMEDIA RM 027 yeast extract
Appendix A. powder as nitrogen source at 20%. The
Twenty-eight (28) g of nutrient pH of the production medium was 7. The
agar was mixed with 1L-distilled water inoculum size was 10% (v/v) of the
in 1L cotton plugged Erlenmeyer flask. production medium. The culture was
The medium was sterilized by incubated at 30°C for 24 h.
autoclaving at 121°C (15 lbs psi) for 15
minutes. 2.6 Optimization of cultural conditions
From the pure culture, cells were
subcultured by streaking them on Factors such as temperature and
nutrient agar plate and incubated at 30°C source of carbon and nitrogen affecting
for 30 h. Several growing colonies were production of amylase were optimized
transferred to nutrient agar slants and by varying parameter one ats a time. The
incubated at 30°C for 18 h. Nutrient agar experiment was conducted in 250 ml
slants were stored in refrigerator and cotton plugged Erlenmeyer flask
subcultured every three (3) weeks. containing 100 ml of production
medium. The pH of 7, temperature at
30°C and 37°C, carbon source at 10%,
2.4 Preparation of inoculum 15%, and 20%, and nitrogen source at
20% were used. The optimum C/N ratio
was determined by varying carbon and
Nutrient broth was used for nitrogen source of production medium.
preparation of inoculum. The medium A factorial design shown below was
contains (in g/L): HIMEDIA RM 027 conducted.
yeast extract, 3; peptone, 5; and NaCl, 8.
The medium was sterilized at 121°C (15 Table 1. Factorial design at pH 7 and
lbs psi) for 15 minutes. 30°C
Three (3) loopfuls of B.
licheniformis 1331 from nutrient agar
slant was inoculated in 100 ml nutrient
broth in 250 ml cotton plugged
Erlenmeyer flask. It was incubated at Carbon source (w/v)
30°C for 18 h. 10% 15% 20% Nitrogen
20% (10%, 20%) (15%, 20%) (20%, 20%) source
(w/v)
2.5 Production medium

The production medium consists
of grinded jackfruit seeds as carbon
source and HIMEDIA RM 027 yeast
extract powder as nitrogen source.
One hundred (100) ml of
production medium in 250 ml
Erlenmeyer flask contains jackfruit seed
as carbon source at 10%, 15%, and 20%
Table 2. Factorial design at pH 7 and The KH2PO4 solution was a
37°C mixture of 0.6803 g of KH2PO4 (0.05M)
and distilled water in 100 ml volumetric
Carbon source (w/v) flask. The NaOH solution was a mixture
of 0.1999 g NaOH and distilled water in
100 ml volumetric flask.
10% 15% 20% One-hundred (100) ml KH2PO4
20% (10%, 20%) (15%, 20%) (20%, 20%) solution was placed in 250 ml beaker
and 10-11 ml of NaOH solution was
The experiment was done in added to have 0.05M KH2PO4-NaOH
triplicate. buffer at pH 6.0.
After determining the optimum
temperature and C/N ratio, 500 ml of 2.8.3 Substrate: Starch solution
production medium in 1L cotton plugged
Erlenmeyer flask was made. Samples 0.2 g soluble starch was
were taken at regular intervals (0, 1, 2, 4, dissolved in boiling 0.05M KH2PO4-
6, 8, 18, 24, 27 and 30h) and analyzed NaOH buffer at pH 6.0 and was cooled
for biomass, protein concentration, and to 40°C.
alpha-amylase activity.
2.8.4 Analytical procedure
2.7 Preparation of crude enzyme
extract For the assay, 1.0 ml of the
diluted enzyme solution (0.5 ml crude
Five (5) ml culture broth in micro enzyme and 0.5 ml buffer) was placed in
test tube was centrifuged for 20 minutes a test tube and warmed to 40°C in a
at 5000 rpm. The supernatant was used water bath for 10 min. Two (2.0 ml) of
as crude enzyme solution to determine starch solution was added and incubated
protein concentration (mg/ml) and alpha- for 10 minutes. Then 0.2 ml was
amylase concentration (units/ml). removed from the test tubes and placed
in another tube containing 5.0 ml of
2.8 Alpha (α)-amylase assay iodine reagent. The absorbance at 620
nm was measured against a blank (0.2
2.8.1 Iodine reagent ml buffer + 5 ml of iodine reagent). The
The stock solution was a mixture substrate control contains 1.0 ml buffer
of two solutions in separate 100 ml + 2 ml substrate in place of diluted
volumetric flask. The two solutions were enzyme solution.
0.5 g iodine in 100 ml water and 5 g α-Amylase activity was
Potassium iodide in 100 ml water. calculated from the absorbances by
The freshly prepared working using the equation:
solution was a mixture of 1 ml stock α-Amylase activity (U/ml)=
solution, 5 ml HCl (5N), and 500 ml Ac − At
deionized water in 500 ml volumetric × 40 D , where: Ac is the
Ac
flask. absorbance of substrate control, At is the
absorbance of test sample, 40 represents
2.8.2 Buffer solution 4.0 mg starch present in the reaction tube
times 10, and D is the enzyme dilution calculating the actual protein
factor. concentration of the enzyme solution.
One unit of alpha-amylase is
defined as the amount of enzyme that
will hydrolyze 0.1 mg of starch in 10 2.10 Biomass determination
min at 40°C when 4.0 mg of starch is
present. Using a pipette, 5 ml of culture
Activities which resulted in broth was placed in pre-weighed micro
absorbances of less than 0.125 after 10 test tubes that have been dried overnight
min required dilution to give linear at 105°C and cooled in a desiccator. The
reactions over the 10-min period (Smith sample was centrifuged for 5 minutes at
and Roe, 1949). 5000 rpm. The supernatant was
discarded. The pellet was resuspended in
2.9 Protein determination by 5 ml distilled water using a vortex mixer.
Bradford assay It was again centrifuged for 5 minutes
and the supernatant was discarded. The
2.9.1 Preparation of Standard micro test tubes with pellets were dried
Curve at 105°C overnight and cooled in a
Twenty (20) µl of each standard desiccator. The sample was weighed and
solution of bovine serum albumin (BSA) the dry cell weight (DCW) of biomass
was prepared at 0 (blank), 125, 250, 500, was calculated.
1000 and 1500 µg/ml. Two (2) ml of
Bradford dye reagent was added to each 3. Results and discussion
solution. The solution was equilibrated
for two minutes to one hour, after which The starch content of jackfruit
the absorbance of each solution was seeds was verified at the Bureau of Plant
measured at 595 nm, using distilled Industry (BPI) before the actual
water as a blank. experiment. The starch content was
37.68% by using Luffshoorl method.
The average of the results of the
2.9.2 Determination of alpha-amylase activity for three trials at
unknown protein content pH 7 and 30°C is shown in Table 5. It
Two (2) ml of Bradford dye can be seen that the highest alpha-
amylase activity was at 10% carbon and
reagent was added to 400 µl of the crude
20% nitrogen.
enzyme solution. The solution was
equilibrated for two minutes to one hour,
Table 3. Alpha-amylase activity at pH 7
after which the absorbance of each
and 30ûC
solution was measured at 595 nm, using
Alpha-amylase activity (U/ml)
distilled water as a blank.
10% Carbon, 20% Nitrogen 0.1592
If the enzyme solution has15% Carbon, 20% Nitrogen 0.059
absorbance reading outside the range20% Carbon, 20% Nitrogen 0.1061
established by the standard curve, the
sample enzyme solution was diluted
with water and absorbance measurement
at 595 nm was redone. Dilution factors
were taken into account when
of the fermentation medium that resulted
0.18
0.16
in the decreased agitation and poor
Alpha-amylase activty (U/ml) 0.14 mixing of air, which were essential for
0.12
0.1
the growth of organism and production
0.08 of enzyme (Haq et. al., 1998). Also,
0.06
0.04
alpha-amylase production was possibly
0.02 affected by the presence of a catabolite
0
1 2 3 repressing carbon source in the growth
% Carbon, % Nitrogen medium.

Figure 1. Alpha-amylase activity at pH Table 5. Protein concentration at pH 7
7 and 30°C and 30°C
Protein concentration (mg/ml)
The average of the results of the 10% Carbon, 20% Nitrogen 3.1338
15% Carbon, 20% Nitrogen 3.3168
alpha-amylase activity for three trials at 20% Carbon, 20% Nitrogen
3.1464
pH 7 and 37°C is shown in Table 4. The
highest alpha-amylase activity was also
at 10% carbon and 20% nitrogen. 3.35

3.3

Table 4. Alpha-amylase assay at pH 7 3.25

and 37ûC 3.2

3.15
Alpha-amylase activity (U/ml)
3.1
10% Carbon, 20% Nitrogen 0.6374 3.05
15% Carbon, 20% Nitrogen 0.609 3

20% Carbon, 20% Nitrogen 0.1658 1 2 3

% C a r bo n , % N i t r o g e n

Figure 3. Protein concentration at pH 7
0.7
0.6
and 30°C
0.5
0.4 Table 6. Protein concentration at pH 7
0.3
0.2
and 37°C
0.1
Protein concentration (mg/ml)
0 10% Carbon, 20% Nitrogen 2.8833
1 2 3 15% Carbon, 20% Nitrogen 2.7915
% Ca r bon, % N i t r og e n 20% Carbon, 20% Nitrogen 3.0993

Figure 2. Alpha-amylase activity at pH 3.2

3.1
7 and 37°C 3

Figure 1 and Figure 2 shows that 2.9

the higher concentration of carbon 2.8

2.7
source has an adverse effect on the
2.6
production of alpha-amylase particularly 1 2 3

at 37°C. It may be due to the thickness % C a r bo n, % N i t r o ge n
Figure 4. Protein concentration at pH 7 and C/N ratio of 10% carbon and 20%
and 37°C nitrogen. Samples were taken at regular
intervals (0, 1, 2, 4, 6, 8, 18, 24, 27, and
The total protein concentration in 30h) for analysis of alpha-amylase
the sample was determined by the activity, protein concentration and
Bradford assay. As shown in Table 5 and biomass (DCW), as shown in Table 7.
Table 6, the highest protein Figure 5 shows the alpha-
concentration was obtained at 30°C, amylase activity in the course of
15% carbon and 20% nitrogen. fermentation as shown in Alpha-amylase
activity was highest during exponential
growth at 4-8 hours and protein
Table 7. Alpha-amylase activity, Protein concentration was highest at 6 hours of
concentration, and Biomass fermentation.
concentration of samples taken at regular
intervals The values of protein
concentration in mg/ml were higher than
Time (h) Alpha-amylase activity (U/ml) Protein concentration (mg/ml) Biomass (mg/ml)
the values of alpha-amylase in U/ml. It
0 0.2879
may2.6988
be due to the fact that0.2
protein
1 0.3165 2.9745 0.26
concentration in jackfruit seeds was
2 0.3275 3.0455 0.2
high.
4 0.4045 3.2888 0.36
6 0.5868 Ajayi
3.3288 (2008) reported
0.38that the
8 0.6484 moisture,
3.224 ash, protein, crude0.3
oil, crude
18 0.6593 fiber, and carbohydrate contents
3.2825 0.22 of
24 0.6593 Artocarpus
3.2975 heterophyllus seeds
0.32 to be
27 0.6659 2.78%, 6.72%, 20.19%, 11.39%,
3.2318 0.12 7.10%,
30 0.6725 and 51.82%, respectively. Bobbio
3.2688 0.38 et al.,
(1978) reported protein, crude lipids, and
carbohydrate contents of jackfruit seeds
3.5 as 31.9%, 1.3%, and 66.2%,
3 respectively. Kumar et al. (1988) also
Alpha-amylase
reported composition of seeds from two
Concentrations (mg/ml)

2.5 activity (U/ml)

2 Protein
varieties of jackfruit. Protein, crude
1.5
concentration
(mg/ml)
lipids, and carbohydrate content were
1
Biomass
concentration
17.8-18.3%, 2.1-2.5%, and 76.1%,
0.5
(mg/ml) respectively. The protein content of
Artocarpus heterophyllus reported by
0
0 10 20 30 40 Bobbio et al. (1978) was very high;
time (h)
however the seeds were reported to have
been collected from fruits of various
Figure 5. Plot of Alpha-amylase varieties of jackfruit.
activity, Protein Concentration, and Table 8. Yields and Productivity
Biomass Concentration during course of
fermentation Time (h) Yamylase/protein Yp/x Productivity
The production of alpha-amylase 0 0.1067 1.4395 N/A
was conducted in 500 ml of medium at 1 0.1064 1.2173 0.3165
pH 7 and optimum temperature of 37°C 2 0.1075 1.6375 0.1637
4 0.123 1.1236 0.1011 Arunava, B., Pal, S.C. and S.K. Sen
6 0.1763 1.5442 0.0978 (1993). Alpha-amylase production in
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