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

In the environment abound
possible sources of essential materials or
substances necessary for the
development or production of certain
products like enzymes, which will be
useful to humans, plants, and other
animals. These substances can be
produced in significantly large amounts
with the advent of biotechnology, but
primarily the materials and the factors
that influence its production source must
be identified. Papain was prepared from
high quality latex of Carica papaya
(Baines, B. S. and Brocklehurst, K.,
1978). Some wastes from fruits and
vegetables can also be a good substrate
for enzyme production. One study was
done on banana fruit stalk, which was
production by Bacillus subtilis under
solid-state fermentation (Krishna and
Chandrasekaran, M., 1996). A similar
study was also done using potato
peelings (Shukla, J. and Kar, R., 2000).
Trypsin/ chymotrypsin inhibitor was
isolated from jackfruit by ammonium
sulfate fractionation and chromatography
(Sai Annapurna, S. and Siva Prasad, D.,
1990). These studies showed that some
enzymes could be produced using
components of fruits and vegetables as
substrate.
This particular study aims to
produce an enzyme by a Bacillus sp.
using the seeds of a locally grown fruit
as substrate. This particular fruit is
jackfruit scientifically known as
Artocarpus heterophyllus or locally
known as “nangka” or “langka”, which
is a favorite dessert of Filipinos and is a
widely grown fruit crop in the
Philippines. It contains carbohydrates,
protein, calcium, iron, sodium,
potassium, B-complex, ascorbic acid,
and small amounts of fats, ash, and iron.
Analysis of jackfruit food composition
per 100 gram edible portion showed that
the carbohydrate content of the seed is
34.90 g (Agriculture and Fisheries
Information Science, Department of
Agriculture).
Based on the above composition,
the jackfruit seed having a high content
of carbohydrate can be used as a
substrate for the production of the
enzyme amylase. The production of
amylase is of great significance in
biotechnology. It is commercially
important in some processing industries
such as beverages, food, textile,
detergents, and paper industries (Pandey
et al., 2000).
This enzyme can be produced
using a specific microorganism or fungi
under controlled conditions. The
amylases can be derived from several
sources ranging from bacteria to plants
to humans. Bacteria and fungi secrete
amylases to the outside of their 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. This particular study will use
Bacillus licheniformis for amylase
production.
This is an exploratory study to
determine alpha-amylase production by
Bacillus licheniformis using jackfruit
seed as substrate. This study is limited to
optimization of cultural and
environmental conditions such as
temperature and carbon/nitrogen ratio.
No enzyme purification and
characterization will be done.
The significance of this study is
that another substrate will be added to
the existing list of carbon sources for
amylase production by microorganisms.
Many industrial processes use waste
materials from plants or animals. Part of
solving the environmental problem of
waste management can be addressed by
the utilization of waste products.
Jackfruit seeds as waste from fruit can be
a potential material for enzyme
production. Being a substrate it will
become a source of income for fruit
vendors and farmers. This study
becomes important to the biotechnology
industry and likewise contributes to
environmental management and
economic stability.
2. Methods
2.1 Preparation of jackfruit seed
One hundred (100) g of jackfruit
seeds was autoclaved in 1L beaker at
121°C (15 lbs psi) for 15 minutes. The
outer seed coating was removed. Seeds
were grinded by using a coffee grinder.
Grinded seeds were placed in zip locked
plastic bag and stored in the freezer.
2.2 Bacterial strain
The bacteria, Bacillus
licheniformis 1331, was obtained from
the Philippine National Collection of
Microorganisms (PNCM), BIOTECH,
University of the Philippines, Los
Baños, Laguna.
2.3 Medium Preparation
HIMEDIA M001 was used for
maintenance of organism. The
composition of nutrient agar is shown in
Appendix A.
Twenty-eight (28) g of nutrient
agar was mixed with 1L-distilled water
in 1L cotton plugged Erlenmeyer flask.
The medium was sterilized by
autoclaving at 121°C (15 lbs psi) for 15
minutes.
From the pure culture, cells were
subcultured by streaking them on
nutrient agar plate and incubated at 30°C
for 30 h. Several growing colonies were
transferred to nutrient agar slants and
incubated at 30°C for 18 h. Nutrient agar
slants were stored in refrigerator and
subcultured every three (3) weeks.
2.4 Preparation of inoculum
Nutrient broth was used for
preparation of inoculum. The medium
contains (in g/L): HIMEDIA RM 027
yeast extract, 3; peptone, 5; and NaCl, 8.
The medium was sterilized at 121°C (15
lbs psi) for 15 minutes.
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 10%
incubated at 30°C for 18 h. 20% (10%, 20%) (15%, 20%) (20%, 20%)
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%
and HIMEDIA RM 027 yeast extract
powder as nitrogen source at 20%. The
pH of the production medium was 7. The
inoculum size was 10% (v/v) of the
production medium. The culture was
incubated at 30°C for 24 h.
2.6 Optimization of cultural conditions
Factors such as temperature and
source of carbon and nitrogen affecting
production of amylase were optimized
by varying parameter one ats a time. The
experiment was conducted in 250 ml
cotton plugged Erlenmeyer flask
containing 100 ml of production
medium. The pH of 7, temperature at
30°C and 37°C, carbon source at 10%,
15%, and 20%, and nitrogen source at
20% were used. The optimum C/N ratio
was determined by varying carbon and
nitrogen source of production medium.
A factorial design shown below was
conducted.
Table 1. Factorial design at pH 7 and
30°C
Carbon source (w/v)
15% 20%
Nitrogen source (w/v)
Table 2. Factorial design at pH 7 and
37°C

Carbon source (w/v)
10% 15% 20%
20% (10%, 20%) (15%, 20%) (20%, 20%)
The experiment was done in
triplicate.
After determining the optimum
temperature and C/N ratio, 500 ml of
production medium in 1L cotton plugged
Erlenmeyer flask was made. Samples
were taken at regular intervals (0, 1, 2, 4,
6, 8, 18, 24, 27 and 30h) and analyzed
for biomass, protein concentration, and
alpha-amylase activity.
2.7 Preparation of crude enzyme
extract
Five (5) ml culture broth in micro
test tube was centrifuged for 20 minutes
at 5000 rpm. The supernatant was used
as crude enzyme solution to determine
protein concentration (mg/ml) and alpha-
amylase concentration (units/ml).
2.8 Alpha (α )-amylase assay
2.8.1 Iodine reagent
The stock solution was a mixture
of two solutions in separate 100 ml
volumetric flask. The two solutions were
0.5 g iodine in 100 ml water and 5 g
Potassium iodide in 100 ml water.
The freshly prepared working
solution was a mixture of 1 ml stock
solution, 5 ml HCl (5N), and 500 ml
deionized water in 500 ml volumetric
flask.
2.8.2 Buffer solution
The KH2PO4 solution was a
mixture of 0.6803 g of KH2PO4 (0.05M)
and distilled water in 100 ml volumetric
flask. The NaOH solution was a mixture
of 0.1999 g NaOH and distilled water in
100 ml volumetric flask.
One-hundred (100) ml KH2PO4
solution was placed in 250 ml beaker
and 10-11 ml of NaOH solution was
added to have 0.05M KH2PO4-NaOH
buffer at pH 6.0.
2.8.3 Substrate: Starch solution
0.2 g soluble starch was
dissolved in boiling 0.05M KH2PO4-
NaOH buffer at pH 6.0 and was cooled
to 40°C.
2.8.4 Analytical procedure
For the assay, 1.0 ml of the
diluted enzyme solution (0.5 ml crude
enzyme and 0.5 ml buffer) was placed in
a test tube and warmed to 40°C in a
water bath for 10 min. Two (2.0 ml) of
starch solution was added and incubated
for 10 minutes. Then 0.2 ml was
removed from the test tubes and placed
in another tube containing 5.0 ml of
iodine reagent. The absorbance at 620
nm was measured against a blank (0.2
ml buffer + 5 ml of iodine reagent). The
substrate control contains 1.0 ml buffer
+ 2 ml substrate in place of diluted
enzyme solution.
α -Amylase activity was
calculated from the absorbances by using
the equation:
α -Amylase activity (U/ml)=
Ac − At
× 40 D , where: Ac is the
Ac
absorbance of substrate control, At is the
absorbance of test sample, 40 represents
4.0 mg starch present in the reaction tube
times 10, and D is the enzyme dilution
factor.
One unit of alpha-amylase is
defined as the amount of enzyme that
will hydrolyze 0.1 mg of starch in 10
min at 40°C when 4.0 mg of starch is
present.
Activities which resulted in
absorbances of less than 0.125 after 10
min required dilution to give linear
reactions over the 10-min period (Smith
and Roe, 1949).
2.9 Protein determination by
Bradford assay
2.9.1 Preparation of Standard
Curve
Twenty (20) µ l of each standard
solution of bovine serum albumin (BSA)
was prepared at 0 (blank), 125, 250, 500,
1000 and 1500 µ g/ml. Two (2) ml of
Bradford dye reagent was added to each
solution. The solution was equilibrated
for two minutes to one hour, after which
the absorbance of each solution was
measured at 595 nm, using distilled
water as a blank.
2.9.2 Determination of
unknown protein content
Two (2) ml of Bradford dye
reagent was added to 400 µ l of the
crude enzyme solution. The solution was
equilibrated for two minutes to one hour,
after which the absorbance of each
solution was measured at 595 nm, using
distilled water as a blank.
10% Carbon, 20% Nitrogen
Ifthe enzyme solution has15% Carbon, 20% Nitrogen
absorbance reading outside the range20% Carbon, 20% Nitrogen
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
calculating the actual protein
concentration of the enzyme solution.
2.10 Biomass determination
Using a pipette, 5 ml of culture
broth was placed in pre-weighed micro
test tubes that have been dried overnight
at 105°C and cooled in a desiccator. The
sample was centrifuged for 5 minutes at
5000 rpm. The supernatant was
discarded. The pellet was resuspended in
5 ml distilled water using a vortex mixer.
It was again centrifuged for 5 minutes
and the supernatant was discarded. The
micro test tubes with pellets were dried
at 105°C overnight and cooled in a
desiccator. The sample was weighed and
the dry cell weight (DCW) of biomass
was calculated.
3. Results and discussion
The starch content of jackfruit
seeds was verified at the Bureau of Plant
Industry (BPI) before the actual
experiment. The starch content was
37.68% by using Luffshoorl method.
The average of the results of the
alpha-amylase activity for three trials at
pH 7 and 30°C is shown in Table 5. It
can be seen that the highest alpha-
amylase activity was at 10% carbon and
20% nitrogen.
Table 3. Alpha-amylase activity at pH 7
and 30˚C
Alpha-amylase activity (U/ml)
0.1592
0.059
0.1061
 the fermentation medium that resulted in
0.18
0.16 the decreased agitation and poor mixing
0.14 of air, which were essential for the
0.12
0.1 growth of organism and production of
0.08
0.06
enzyme (Haq et. al., 1998). Also, alpha-
0.04 amylase production was possibly
0.02
0
affected by the presence of a catabolite
1 2 3 repressing carbon source in the growth
% C arbon, % 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)
10% Carbon, 20% Nitrogen 3.1338
The average of the results of the
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 37˚C 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 arbon, % Nitrogen

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
% C arbon, % Nitrogen
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 of % C arbon, % Nitrogen

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, 15% amylase activity in the course of
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)
the values of alpha-amylase in U/ml. It
0 0.2879
1 0.3165
may be due to the fact that protein
2 0.3275 concentration in jackfruit seeds was
4 0.4045 high.
6 0.5868 Ajayi (2008) reported that the
8 0.6484 moisture, ash, protein, crude oil, crude
18 0.6593
fiber, and carbohydrate contents of
24 0.6593
Artocarpus heterophyllus seeds to be
27 0.6659
30 0.6725
2.78%, 6.72%, 20.19%, 11.39%, 7.10%,
and 51.82%, respectively. Bobbio et al.,
(1978) reported protein, crude lipids, and
3.5
carbohydrate contents of jackfruit seeds
3
as 31.9%, 1.3%, and 66.2%,
Alpha-
am ylase
activity
respectively. Kumar et al. (1988) also
2.5
(U/m l)
Protein
reported composition of seeds from two
varieties of jackfruit. Protein, crude
2
concentration
(mg/m l)
1.5

Biomass
lipids, and carbohydrate content were
1 concentration
(mg/ml)
17.8-18.3%, 2.1-2.5%, and 76.1%,
0.5
respectively. The protein content of
0
0 20 40
Artocarpus heterophyllus reported by
ti me (h) Bobbio et al. (1978) was very high;
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
Biomass Concentration during course of Table 8. Yields and Productivity
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
6 0.1763 1.5442
8 0.2011 2.1613
18 0.2009 2.9968
24 0.2 2.0603
27 0.2061 5.5492
30 0.2057 1.7697
Table 8 shows the yields and
productivity in the course of
fermentation. It can be seen that alpha-
amylase production was highest during
exponential growth and was almost
complete during the stationary phase.
4. Conclusion
Alpha-amylase was successfully
produced using jackfruit seed as solid
substrate having a starch content of
37.68% in 100 grams seeds. The
optimum conditions for alpha-amylase
production were pH of 7, temperature of
37°C and C/N ratio of 1:2. Alpha-
amylase activity was highest during
exponential growth at 4-8 hours of
fermentation. Protein concentration was
highest at 6 hours of fermentation.
References
Agriculture and Fisheries Information
Science, Department of Agriculture,
Philippines.
Ajayi, I. A. (2008). Comparative study
of the chemical composition and mineral
element content of Artocarpus
heterophyllus and Treculia africana
seeds and seed oils. Bioresource
Technology, Volume 99, 5125-5129.
Anyangwa, E. M., Mapsev, C.,
Musanage, P. and M. Elemva (1993).
The effect and removal of starch in the
sugar refining industry. J. Int. Sugar.,
Volume 95, 210-213.
0.1011 Arunava, B., Pal, S.C. and S.K. Sen
0.0978 (1993). Alpha-amylase production in
0.081 lactose medium by Bacillus circulanse.
0.0366 J. Microbiologia., Volume 9 (2),142-
0.0274 148.
0.0247
0.0224
Awal, H. M. A. and S. Gheyasuddin
(1991). Biochemical parameters of
jackfruit seed-meal. Bangladesh Journal
of Agricultural Research, Volume 16
(1), 17-22.
Baines, B. S. and K. Brocklehurst
(1979). A Necessary Modification to the
Preparation of Papain from Any High-
Quality Latex of Carica papaya and
Evidence for the Structural Integrity of
the Enzyme Produced by Traditional
Methods. Biochem J., Volume 177 (2),
541-548.
Bhat, A. V. and T. N. Pattabiraman
(1989). Protease inhibitor from jackfruit
seed (Artocarpus integrifolia). Journal
of Biosciences, Volume 14 (4), 351-365.
Bernfeld, P. (1955). Amylases, α , β ,
Methods in Enzymology. Volume 1,
149-155.
Bobbio, F. O., El-Dash, A. A., Bobbio,
P. A., and L. R. Rodrigues (1978).
Isolation and characterization of the
physico-chemical properties of the starch
of jackfruit seed (Artocarpus
heterophyllus). Cereal Chem, Volume
55, 501-511.
Borchet T.V.F., Lassen, S. E., Svendsen,
A. and H.B. Frantzen (1995). Oxidation
stable amylase for detergent. J.
Biotechnol., Volume 10,175-179.
Burhan A., Nisa U., Gokhan C., Omer
C., Ashabil A., Osman G. (2003).
Enzymatic properties of a novel
thermostable, thermophilic, alkaline, and
chelator resistant amylase from an
alkaliphilic Bacillus sp. Isolate ANT-6.
Process Biochem, Volume 38 (10),
1397-1403.
Castro, G. R., Biagori, M. D. and F.
Sineriz (1999). Studies on alpha-amylase
production by Bacillus licheniformis
MIR-61. Acta. Biotechnol., Volume 19
(3), 263-272.
De-Cordt, S., Vanhoof, K., Hu, J.,
Maesmans, G., and P. Tobback (1994).
The influence of polyalcohols and
carbohydrate on the thermostability of
alpha-amylase. J. Bioeng., Volume 43
(2), 107-114.
de Oliveira A. N., de Oliveira L. A.,
Andrade J. S., and A. F. C. Junior
(2007). Rhizobia amylase production
using various starchy substances as
carbon substrates. Brazilian Journal of
Microbiology, Volume 38, 208-216.
Dharani Aiyer, P. V. (2004). Effect of
C:N ratio on alpha-amylase production
by Bacillus licheniformis SPT 27.
African Journal of Biotechnology,
Volume 3 (10), 519-522.
Ednord, R. and K. Dietrich (1996).
Kinetics of starch hydrolysis with
Bacillus amyloliquefaciens alpha-
amylase under high hydrostatic pressure.
J. Biotechnol., Volume 48 (11-12), 409-
414.
Emanuilova, E. I. and K. Toda (1984).
Alpha-amylase production in batch and
continuous culture by Bacillus
caldolyticus. Appl. Microbiol.
Biotechnol., Volume 19, 301-305.
Ekunsaumi, T. Laboratory Production
and Assay of Amylase by Fungi and
Bacteria. UW-Washington country.
Fukumoto, J., Yamamoto, T., and K.
Ichikawa (1951). Crystallization of
bacterial saccharogenic amylase and the
properties of the cyrstalline amylase.
Proc. Jap. Acad., Volume 27, 352-358.
Fuwa, H. (1954). A new method for
microdetermination of amylase activity
by the use of amylose as the substrate. J.
Biochem., Volume 41, 583-603.
Harger, C., Sprada, D., and E. Hiratsuka
(1982). Amilase Fungica. In:
Bioquimica das Fermentacoes. 56.
Haq, I., Ashraf, H., Zahara, R., and M.
A. Qadeer (1998). Biosynthesis of alpha-
amylase by Bacillus subtilis GCB-12
using agricultural by products as
substrates. Biologia, Volume 44 (1&2),
154-163.
Haq, I., Ashraf, H., Zahara, R., and M.
A. Qadeer (2003). Production of alpha-
amylase by Bacillus licheniformis using
an economical medium. Bioresource
Technology , Volume 87, 57-61.
Haq, I., Ashraf, H., Iqbal, J.., and M. A.
Qadeer (2005). Pearl millet, a source of
alpha-amylase production by Bacillus
licheniformis. Bioresource Technology ,
Volume 96, 1201-1204.
Hendrickx, M., Tobback, P., Avila, I.,
and S. Cordt (1994). DSC and protein-
based time-temperature integrators: Case
study of alpha-amylase stabilized by
polyols and/or sugar. Biotechnol.
Bioeng., Volume 4 (7), 859-865.
Ivanova, V., Yankov, D., Kabaivanova,
L. and D. Pashkkoulov (2001).
Simultaneous biosynthesis and
purification of two extra cellular Bacillus
hydrolases in aqueous two alpha-
amylase. J. Biochem. Eng., Volume 8
(1), 61-81.
Kathiresan K. and S. Manivannan
(2006). Alpha-amylase production by
Penicillium fellutanum isolated from
mangrove rhizosphere soil. African
Journal of Biotechnology Volume 5
(10), 829-832.
Khajeh, K., Naderi, H., Ranjbar, B.,
Moosavi, A., and M. Nemat (2001).
Chemical modification of lysine residues
in Bacillus alpha-amylases: Effect on
activity and stability. Enzyme.
Microbiol. Technol., Volume 28 (6),
543-549.
Krishnan, T. and A. K. Chandra (1982).
Effect of oilseed cakes on alpha-amylase
production by Bacillus licheniformis
CUMC 305. Appl. Environ. Microbiol.,
Volume 44 (2), 270-274.
Krishnan, T. and A. K. Chandra (1983).
Correlation between alpha-amylase
production and sporulation in Bacillus
licheniformis CUMC 305 with respect to
the effect of some carbohydrates and
phenylmethylsulfonyl flouride treatment.
Zentralbl Mikrobiol., Volume 138 (6),
475-485.
Kumar, S., Singh, A. B., Abidi, A. B.,
Upadhyah, R. G., and A. Singh (1988).
Proximate composition of jackfruit
seeds. J. Food Sci. Tech, Volume 25,
308-309.
Madigan, Michael T., Martinko, John
M., and Jack Parker (2003). Brock
Biology of Microorganisms. 10th Edition.
Pearson Education, Inc. 980-981.
Nickless, D. M., Sobieski, R. J., and S.
S. Crupper (2001). Genetic Regulation
of Amylase Expression in Bacillus.
Bioscene, Volume 27 (4), 27-29.
Niziolek S. (1998). Production of
extracellular amylolytic enzymes by
some species of the genus Bacillus.
Acta. Microbiologica. Polonica.,
Volume 47 (1), 19-29.
Nomura, M., Moruo, B., and S. Akabor
(1956). Studies on amylase fermentation
by Bacillus subtilis. Effect of high
concentration of polyetylene glycol on
amylase formation by Bacillus subtilis.
J. Biochem., Volume 43, 143.
Odoemalam, S. A. (2005). Functional
Properties of Raw and Heat Processed
Jackfruit (Artocarpus heterophyllus).
Pakistan Journal of Nutrition, Volume 4
(6), 366-370.
Onyeike, E. N., Olungwe, T., and A. A.
Uwakwe (1995). Effect of heat treatment
and defatting on the proximate
composition of some Nigerian local soup
thickeners. Food Chem, 53, 173-175.
Padmanabhan, S., Ramakrishna, M.,
Lonsane, B.K., and M. M. Krishnaiah
(1992). Enhanced leaching of product at
elevated temperatures: Alpha-amylase
produced by Bacillus licheniformis M27
in solid state fermentation system. Lett.
Appl. Microbiol., Volume 15 (6), 235-
238.
Pandey, A. (1990). Aspects of Fermenter
Design for Solid State Fermentation,
Process Biochemistry. Volume 26, 335-
361.

Pandey, A., Nigam P., Soccol C. R.,
Soccol, V. T., Singh D., and R. Mohan
(2000). Advances in microbial amylases.
Biotechnol. Appl. Biochem, Volume 31,
135-152.
Pandey, A., Webb, C., Soccol, C. R., and
C. Larroche (2005). Enzyme
Technology. New Delhi: Asiatech
Publishers, Inc. 197.
Pretorius, S., De-Kock, M. J. Britz, T. J.,
Potgieter, H. J. and P. M. Lategan
(1986). Numerical taxonomy of alpha-
amylase producing strain of Bacillus
species. J. Appl. Bacteriol., Volume 60
(4), 351-360.
Pratima, B. and Umender, S. (1989).
Production of alpha-amylase in a low
cost medium by Bacillus licheniformis
TCRC-B13. J. Ferment. Bio. Eng.,
Volume 67 (6), 422-423.
Ramesh, M. V. and B. K. Lonsane
(1990). Critical importance of moisture
content of the medium in alpha-amylase
production by Bacillus licheniformis
M27 in a solid-state fermentation
system. Appl. Microbiol, Biotechnol.,
Volume 33 (5), 501-505.
Ramesh, M.V. and B. K. Lonsane
(1991). Ability of solid state
fermentation technique to significantly
minimize catabolic repression of alpha-
amylase production by Bacillus
licheniformis M26. Appl. Microbiol.
Biotechnol., Volume 35 (5), 591-593.
Rehana, F., Venkatasubbaiah, P. and K.
Nand (1989). Preliminary studies on the
production of thermostable alpha-
amylase by a mesophilic strain of
Bacillus licheniformis. Chem. Mikrobiol.
Technol. Lebensem., Volume 12 (1), 8-
13.
Rothstein, D. M., Devlin, P. E., and R.
L. Cate (1986). Expression of alpha-
amylase in Bacillus licheniformis.
American Society for Microbiology,
Volume 168 (2), 839-842.
Sai Annapurna S. and D. Siva Prasad
(1991). Purification of trypsin/
chymotrypsin inhibitor from jackfruit
seeds. Journal of the Science of Food
and Agriculture, Volume 54 (3), 399-
411.
Saito N. and K. Yamamoto (1975).
Regulatory factors affecting alpha-
amylase production in Bacillus
licheniformis. American Society for
Microbiology, Volume 121 (3), 848-856.
Shukla J. and R. Kar (2006). Potato peel
as a solid substrate for thermostable
alpha-amylase production by
thermophilic Bacillus isolates. World
Journal of Microbiology and
Biotechnology, 417-422.
Smith, B. W. and J. H. Roe (1949). A
photometric method for the
determination of alpha-amylase in blood
and urine with the use of the starch-
iodine color. J. Biol. Chem., Volume
179, 53-56.
Spier, M. R., Vandenberghe L. P. D. S.,
Woiciehowski A. L., C. R. Soccol
(2006). Production and Characterization
of Amylases by Aspergillus niger under
Solid State Fermentation Using Agro
Industrial Products. International
Journal of Food Engineering, Volume 2
(3), 6.
Tulyathan V., Tananuwong K.,
Songjinda P., and N. Jaiboon (2002).
Some Physicochemical Properties of
Jackfruit (Artocarpus heterophyllus
Lam) Seed Flour and Starch. Science
Asia, Volume 28, 37-41.

Vortruba, J., Emanuilova, E.,

Kaymakchiev, A. and J. Pazlarova
(1984). Kinetics of alpha-amylase
production in a continuous culture of
Bacillus licheniformis. Folia. Microbiol.,
Volume 29 (1), 19-22.

Weemaes, C., De-Cordt, S., Goossens,

K., Ludikhuyze, L., Hedrickx, M.,
Heremans, K., and P. Tobback (1996).
High pressure, thermal, and combined
pressure temperature stabilities of alpha-
amylases from Bacillus species.
Biotechnol. Bioeng. Volume 50 (1), 49-
56.

Wilson, J. J. and W. M. Ingledew

(1982). Isolation and characterization of
Schwanniomyces alluvius amylolytic
enzymes. Appl. Env. Microbiol., Volume
44, 301-307.