Alpha-amylase Production by Bacillus licheniformis

1331 using Jackfruit Seeds as Substrate

by

Rica Mae A. Palongpalong

An Undergraduate Thesis Submitted to the School of

Chemical Engineering and Chemistry
in Partial Fulfillment of the Requirements for the Degree

Bachelor of Science in Biotechnology

Mapúa Institute of Technology

December 2008
ii
 APPROVAL SHEET

This is to certify that we have supervised the preparation of and read the thesis / practicum or
research report prepared by Rica Mae A. Palongpalong entitled Alpha-amylase
Production by Bacillus licheniformis 1331 using Jackfruit Seeds as Substrate and that the
said thesis / practicum or research report has been submitted for final examination by the
Oral Examination Committee.

Percival G. Garcia Flordeliza C. De Vera

Adviser Course Adviser

As members of the Oral Examination Committee, we certify that we have examined this
report and hereby recommend that it be accepted as a fulfillment of the practicum for the
Degree Bachelor of Science in Biotechnology.

Kevin B. Dagbay Arturo L. Tapas Jr.

Panel Member 1 Panel Member 2

Russell S. Julian
Panel Member 3

This practicum paper is hereby approved and accepted by the School of Chemical
Engineering and Chemistry as fulfillment of the practicum requirement for the Degree
Bachelor of Science in Biotechnology.

Isidro C. Medina, Jr. Luz L. Lozano

Head (OIC), Dean
Biotechnology Program

iii
 ACKNOWLEDGEMENT

I would like to express my sincere gratitude to the following people who have
contributed in the completion of my thesis:

First, to God Almighty, for all the countless blessings and graces through the following
people who have been instrumental in the completion of my thesis.

To my mother, Dr. Beauty A. Palongpalong, for all the support that she has given me.
Thank you for the encouragement, the sleepless nights of coaching and correcting my
sentence construction, for pushing me hard to sustain my spirit to reach the finish line. Most
of all the friendship, unconditional love, care, and for nurturing me to be the person that I am
today.

To my late father, Dr. Artemio D. Palongpalong, PhD. whose memory has served as my
inspiration to finish my thesis, he being a professor and Dean of the prime university of the
country.

To my uncle and aunt, Engr. Ruel Janolino and Grace Janolino, for being my second
father and mother respectively. Thank you so much for the financial support, paying my
tuition fees, giving my daily allowance and other personal needs, for lending me the coffee
grinder for grinding the jackfruit seeds and the money for the chemicals used in the study.
Most of all the strong moral support and encouragement and cannot be overemphasize is the
pampering just like a spoiled brat kid. The gesture of treating me like your own baby girl will
stay in my heart forever.

To my brother, Heinrich Victor Valery A. Palongpalong for being understanding that he

gives way every time I need the computer for my thesis.

To Prof. Percival G. Garcia, my very supportive thesis adviser, for initially approving my
thesis topic, purchasing the bacteria from UPLB, patiently checking the English grammar,
giving me protocols for the methodology, help me analyze the data and for all the advises,
guidance, and continuous support will not be forgotten.

To Prof. Kevin B. Dagbay, Prof Russell S. Julian, and Dr. Arturo L. Tapas Jr. my
respective panellists, for sharing their ideas, screening my thesis paper, their objective
criticisms and suggestions on the improvement of the study. Thank you so much.

To Prof. Herbert J. Santos and Prof. Flordeliza C. De Vera, my thesis 1 and thesis 3
course coordinator respectively, for sharing suggestions and ideas regarding the study.

To Engr. Ariziel Ruth Marquez, Former Head of Advance Chemistry Laboratory, for
giving me permission to use the UV-Vis spectrophotometer and analytical balance.

To Ate Lolit, for helping me operate the UV-Vis spectrophotometer in Physical Chemisty
Laboratory.

iv
To Ate Thelma, for helping me make the KH2PO4-NaOH buffer for my alpha-amylase assay
and for the friendship we had since I was a freshman in Mapua.

To the Mapua guards, for ensuring our safety 24 hours a day when we did our laboratory
work overtime.

To Jerico Jayson D.C. Uy, for helping me in doing the culture of my bacteria, doing the
preparation of standard curve for Bradford assay, and assisting me in my experiment.

To Jansen Jan T. Chua, for helping me also in doing the culture of my bacteria

To Edison I. Pineda, for writing the letter allowing us to use the N313 or Microbiology
Laboratory overnight.

To Kathrina R. Siongco, for helping me make the combined graph of Alpha-amylase assay,
Bradford assay, and Biomass versus time in hours.

To Biologic and Chemsoc family, for being part of your family and letting me serve my two
organizations as an Executive Secretary for Chemsoc or Chemistry Society of Mapua and
Assistant Secretary and Secretary for Biologic, Association of Biotechnology students of
Mapua. You have inspired me to go on up to the final stage of my stay in Mapua.

To UPIS Batch 2004, for the friendship, bonding, and unity that we had since our
Kindergarten days, partly you have encouraged me during my lowest points in Mapua.

To Elaine L. Cunanan, for being my best friend since freshmen through thick and thin,
through highs and lows, for always being there for me, and for your loyalty.

To Aldrich Thomas R. Agtarap and Alberto L. Abadiano Jr., for being such a good and
loyal friend, and for being always ready to lend your helping hand in times of my needs.

To those whom I forgot to mention, thank you for everything.

For this achievement, I give back all the glory and praises to the omnipotent Father
Almighty.

Rica Mae A. Palongpalong

v
 TABLE OF CONTENTS

TITLE PAGE i

APPROVAL PAGE ii

ACKNOWLEDGEMENT iii

TABLE OF CONTENTS v

LIST OF TABLES vii

LIST OF FIGURES viii

ABSTRACT ix

Chapter 1: INTRODUCTION 1

Chapter 2: REVIEW OF LITERATURE 3

Jackfruit seed 3

Amylase 6

Alpha (α )-amylase 7

Alpha (α )-amylase Production 8

Chapter 3: ALPHA-AMYLASE PRODUCTION BY BACILLUS LICHENIFORMIS

1331 USING JACKFRUIT SEEDS AS SUBSTRATE 21

Abstract 21

Introduction 21

Methodology 23

Preparation of jackfruit seed 23

Bacterial Strain 23

Medium Preparation 24

Preparation of inoculum 24

vi
 Optimization of cultural conditions 25

Preparation of crude extract enzyme 26

Alpha (α )-amylase assay 26

Iodine reagent 26

Buffer solution 26

Substrate: Starch solution 26

Analytical procedure 26

Bradford assay 27

Preparation of Standard Curve 27

Determination of Unknown Protein Content 28

Biomass determination 28

Results and Discussion 29

Conclusion 36

Recommendation 36

References 36

Chapter 4: CONCLUSION 42

Chapter 5: RECOMMENDATION 43

REFERENCES 44

APPENDICES 49

A: Composition of HIMEDIA M001 50

B: Assay on Alpha-amylase Activity 51

C: Assay on Protein Concentration 52

D: Alpha-amylase activity, Protein Concentration, and Biomass Concentration

of Samples Taken at Regular Intervals 54

vii
 LIST OF TABLES

Table 1: Factorial design at pH 7 and 30°C 25

Table 2: Factorial design at pH 7 and 37°C 25

Table 3: Alpha-amylase activity at pH 7 and 30°C 29

Table 4: Alpha-amylase activity at pH 7 and 37°C 30

Table 5: Protein concentration at pH 7 and 30°C 31

Table 6: Protein concentration at pH 7 and 37°C 32

Table 7: Alpha-amylase activity, Protein Concentration, and Biomass Concentration of

samples taken at regular intervals 33

Table 8: Yields and Productivity 35

Table A.1: Composition of HIMEDIA M001 45

Table B.1: Alpha-amylase activity at pH 7 and 30°C 46

Table B.2: Alpha-amylase activity at pH 7 and 37°C 46

Table C.1: Tabulation of absorbance versus BSA concentration 47

Table C.2: Protein concentration at pH 7 and 30°C 48
Table C.3: Protein concentration at pH 7 and 37°C 48

Table D.1: Alpha-amylase activity of samples taken at regular intervals 49

Table D.2: Protein concentration of samples taken at regular intervals 50
Table D.3: Biomass concentration of samples taken at regular intervals 52
Table D.4: Specific enzyme activity (Yamylase/protein), Yp/x, and Productivity of samples
taken at regular intervals

viii
 LIST OF FIGURES

Figure 1: Alpha-amylase activity at pH 7 and 30°C 29

Figure 2: Alpha-amylase activity at pH 7 and 37°C 30

Figure 3: Protein concentration at pH 7 and 30°C 31

Figure 4: Protein concentration at pH 7 and 37°C 32

Figure 5: Plot of Alpha-amylase activity, Protein Concentration, and Biomass

Concentration during course of fermentation 33

Figure C.1: Plot of BSA standard curve 47

Figure D.1: Plot of Alpha-amylase activity of samples taken at regular intervals 49

Figure D.2: Plot of Protein concentration of samples taken at regular intervals 50
Figure D.3: Plot of Biomass concentration of samples taken at regular intervals 51

ix
 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

x
 Chapter 1

INTRODUCTION

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 used as a substrate for alpha-amylase 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).

1
 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
 Chapter 2

REVIEW OF LITERATURE

Almost all of the chemical changes brought about by the cells of all living

organisms such as animals, plants, and microorganisms are mediated by appropriate catalyst.

Enzymes are the protein biocatalysts produced by living cells. Considerable quantities of

enzymes are currently produced commercially from animal and plant sources. To produce

such enzymes there must be a substrate as a carbon source and a nitrogen source processed

under controlled conditions. This study identified jackfruit seed as a substrate to make seeds

useful since these are thrown away after eating the pulp.

Jackfruit seed

Several studies about jackfruit were done in Asia particularly in South and

Southwest Asia and South America. S. Sai Annapurma and D. Siva Prasad (1991) made a

study on the purification of trypsin/chymotrypsin inhibitor, which was isolated from jackfruit

seeds, by ammonium sulfate fractination and chromatography on DEAE cellulose and

SephadexG-100. During all stages of purification the ratio of trypsin and chymotrypsin

inhibitory activities remained constant. Vanna Tulyathan et al. (2002) of Chulalongkorn

University in Thailand did some investigation studies on physicochemical properties of

jackfruit seed flour and starch. Flour from jackfruit has high protein and carbohydrate

content. It also has good water and oil absorption abilities. From the result of the study,

amylose content of jackfruit seed starch was 32% and is higher than tapioca starch (17%) and

corn starch (26%). These starchy substances can be used as substrate for carbon, a necessary

element for the production of amylase by Bacillus. Jackfruit can be a potential source of

3
carbon in amylase production since it has a high protein and carbohydrate content (Tulyathan

et al., 2001). A study on the factorial design evaluation of some experimental factors for

phenol oxidation using crude extracts from jackfruit was published in the Journal of the

Brazilian Chemical Society (2002).

Another study involving jackfruit seed was conducted by Bhat AV and Pattabiraman

(1989) where the protease inhibitory activity of jackfruit seed (Artocarpus integrifolia) was

investigated. The inhibitory with a molecular weight of 26 kilodalton was found to be a

glycoprotein. Galactose, glucose, mannose, fructose, xylose, glucosamine, and uronic acid

were identified as constituent units of the inhibitor. Dansylation and electrophoresis in the

presence of mercaptoethanol indicated that the inhibitor is made up of more than one

polypeptide chain. It powerfully inhibited the caseinolytic activities of rabbit and horse

pancreatic preparations and was least effective on human and pig pancreatic extracts.

It was published in Bangladesh Journal of Agricultural Research (1991) a

biochemical study on jackfruit seed meal that reveals its proximate composition, soluble

protein distribution pattern, peptisation, starch pattern and mineral composition. The meal

was found to contain 7.44% moisture, 1.86% ash, 2.88% crude fiber, 1.48% lipids, 75.03%

carbohydrate, and 11.31% crude protein. Protein distribution pattern according to Osborn

scheme revealed that the major protein fractions were albumin and glutelin, which together

constituted 54.73% of the extracted protein. Globulin and prolamin were the minor fractions,

which together comprised 6.63% of the extracted protein. A considerable percentage of

protein (38.64%) could not be extracted by the solvents used. Peptisation of nitrogen as a

function of pH in water revealed that the maximum extraction of 73.47% was obtained at pH

13, while the minimum extraction of 11.55% at pH 3. The meal contained 60.7% starch

4
consisting of 56.29% amylose and 43.71% amylopectin, their ratio being 100:78. Starch

granules were oval in shape. Mineral composition of the meal were P(0.13%), Ca (0.59%),

Mg (0.41%), K(0.45%), S(0.15%), B(0.08%), Fe (120 ppm), Cu(50 ppm), Mn (1.47 ppm)

and Zn (56 ppm). Evidently, jackfruit seed meal can be used as ingredient for the products

that require easy dispersion.

There were several studies on the properties of jackfruit seed flour. One of which

was done by S.A. Odoemelam (2005) where functional properties of raw and heat processed

jackfruit flour were determined. The functional properties, water and oil absorption, gelation,

bulk density, foaming, emulsification, and nitrogen solubility were studied. The effects of pH

and NaCl concentration on some of these functional properties were also investigated.

Emulsification, foam capacity, and nitrogen solubility were pH dependent with minimum

values at pH 4. Addition of NaCl up to 0.4 M improved emulsification capacity of both raw

and heat processed flours whereas a decrease was observed in foam capacity at 0.2 M. The

foam of the raw flour was more stable than that of the heat processed flour. Heat processing

of jackfruit flour increased the water and oil absorption capacity but lowered nitrogen

solubility, foam capacity, and emulsification capacity. Water and oil absorption capacities of

raw jackfruit flour were 2.3 ml/g and 2.8 ml/g, respectively, while heat processed flour

sample gave 3.5 ml/g and 3.1 ml/g. The water and oil absorption capacities of the heat

processed jackfruit flour were significantly higher (p<0.05) than those of the raw flour. Least

gelation concentration of raw jackfruit was found to be 16% and heat processed flour, 18%

while bulk densities of 0.61 g/ml were calculated for raw and heat processed flours.

A comparative study of the chemical composition and mineral element content of

jackfruit and breadfruit seeds and seed oils was conducted by Ibironke Adetolu Ajayi (2006).

5
The two seeds from Moraceae family, jackfruit and breadfruit are quite rich in oil, protein,

carbohydrate, and some mineral elements. The oil content of the seeds classifies them as

average oil yielding. Potassium is high in jackfruit seed as compared to breadfruit followed

by sodium, magnesium, calcium, iron, zinc, and copper in descending order.

The previous studies showed that there were existing researches done involving

jackfruit. This particular study on alpha-amylase production using jackfruit as a substrate is

new and relatively unexplored study in the Philippines.

Amylase

Enzymes are named according to the substrate on which they act, therefore, the term

amylase indicates action on starch, which contains two types of polysaccharides: 15- 20% of

amylose and 80-85% of amylopectin (Harger, 1982). Gupta et al. (2003) and Pandey et al.

(2005) described that amylase catalyzes hydrolysis of starch molecules liberating diverse

products, including dextrins and progressively, small glucose polymers.

Amylases are classified according to how it break down starch molecules. Alpha-

amylase reduces the viscosity of starch by breaking down the bonds at random producing

varied sized chains of glucose. Beta-amylase breaks the glucose-glucose bonds down by

removing two glucose units at a time, thereby producing maltose. The third type is the

amyloglucosidase, which breaks successive bonds from the non-reducing end of the straight

chain, producing glucose. Bacteria and fungi secrete amylases to the outside of their cells to

carry out extracellular digestion. When the insoluble starches are broken down, the soluble

end products such as glucose or maltose are absorbed into the cells of the organisms. These

enzymes present great importance in biotechnology with applications in the processing of

fermented beverages, food, and additives to detergents for removing stains, saccharification

6
of starch for alcohol production, brewing, textile industries, and paper industries. Despite

being able to be extracted from diverse sources, including plants, animals, and

microorganisms, microbial enzymes generally find great industrial demand. A large number

of microbial amylases are available commercially and have almost completely replaced

chemical hydrolysis of starch in starch processing industry (Pandey et al., 2000). Amylases

are among the most important enzymes used in biotechnology, particularly in process

involving starch hydrolysis. Though amylases originate from different sources like plants,

animals, and microorganisms, the microbial amylases are the most produced and used in

industry, due to their productivity and thermostability (Burhan et al., 2003).

Alpha (α )-amylase

Pandey et al., (2005) define α -amylase (1,4-α -glucan-4-glucanohydrolase, EC

3.2.1.1.) as an enzyme that breaks the α (1,4) bonds of polysaccharides that have ten or more

units of D-glucose united by α -1,4 bonds. The attack occurs in a non-selective form (as

endoenzyme) on different points of the chain simultaneously, so that the first hydrolysis

products are oligosaccharides of 5 to 7 units of glucose. This represents a preferential attack

on each step of the helix, of the amylose or amylopectin spiral chain. The α -amylase

cleaving point, which after its attack on the represented links, originate fragments of 5 to 7

units of glucose. After hydrolysis, units of glucose, oligosaccharides of different molecular

weights and dextrins are released. It acts, isolatedly or simultaneously, with other amylolytic

enzymes, presenting important applications in the food, drinks, textile, and pharmaceutical

industries. Endogenous α -amylase of cereal seeds are used in baking and beer industry,

while of microbial enzymes are used in processes that require saccharification and

liquefaction of starch.

7
Alpha (α )-amylase Production

Bacterial amylase could be produced by Bacillus species, Pseudomonas,

Saccharophila, and Clostridium species. But on industrial scale the strains of Bacillus species

seem to be preferred. Fukumoto (1951) carried out extensive work on the production of

alpha-amylase by Bacillus species. Later on, many other scientists attempted towards the

production of alpha-amylase by Bacillus species.

Pretorius et al. (1986) have isolated 134 alpha-amylase producing strains of Bacillus.

The strains were divided into 12 groups and their biochemical and morphological

characterizations were carried out. The isolates were related to Bacillus subtilis, Bacillus

licheniformis, and Bacillus amyloliquefaciens.

Using enrichment techniques a large number of microorganisms were isolated and

their alpha-amylase activity was detected by flooding the plates with a weak iodine solution.

Several alpha-amylase producing strains of yeast, fungi, and actinomycetes were isolated.

Among them a mesophilic strain of Bacillus licheniformis B-29 produced the maximum

enzyme activity extracellularly in submerged conditions. The productivity of enzyme was

optimum at pH 7.0 after 72 h of cultivation with 2% rice slurry or sorghum in the medium

(Rehana et al., 1989).

Borchet et al. (1995) stated that the addition of amylase to detergent would be

possible means for the removal of starch strains. The Bacillus licheniformis was chosen as

the most suitable source of alpha-amylase for this purpose. Ednord and Dietrich (1996)

observed that Bacillus licheniformis showed high capacity for the production of alpha-

amylase. The resulting enzyme preparation was stabilized by CaCl2 at concentration as low

as 10-30 ppm and was active at temperature up to 98°C.

8
 Niziolek (1998) have studied the production of extracellular amylolytic enzymes in

41 strains of the genus Bacillus representing 13 species using different liquid media and

cultivation temperature of 30°C and 38°C. It was found that 8 strains were amylase-negative,

19 strains were low-productive and 12 were medium-productive strains (10-25 U/ml).

Bacillus subtilis AS-1-108, Bacillus subtilis NCIB 8159, and Bacillus licheniformis NCIB

7198 strains were included among the higher-productive as they produced about 370, 170,

and 40 U/ml of alpha-amylase, respectively. The enzymes from Bacillus subtilis AS-1-108

and NCIB 8159 strains were more thermosensitive than those of the medium-productive

strains of Bacillus subtilis.

An acid stable alpha-amylase hyperproducing strain, designated as MIR-61, was

isolated in a screening procedure from South American soil samples. MIR-61, a 60°C

thermo-resistant strain, was identified using 98 biochemical and morphological tests and

characterized as Bacillus licheniformis by numerical taxonomy. Batch cultures of Bacillus

licheniformis MIR-61 showed extracellular alpha-amylase and alpha-glucosidase activities

during exponential growth phase. The production of alpha-amylase was studied at constant

pH values at 37°C and 45°C. Maximum alpha-amylase activity (4,767 kU/dm3 in a liquid

medium) was detected at 45°C at a constant pH 7.0 in the late exponential phase. The alpha-

amylase production of Bacillus licheniformis MIR-61 was 10 to 300 times higher than the

enzyme production reported in strains of the same species. Optimum alpha-amylase activity

was found at 50 to 67°C in an acid pH range from 5.5 to 6.0 (Castro et al., 1999).

Khajeh et al. (2001a) made the comparative study on limited and extensive

proteolysis of mesophilic alpha-amylase from Bacillus amyloliquefaciens (BAA) and

thermophilic from Bacillus licheniformis (BLA) using trypsin. As expected, the thermophilic

9
enzyme showed greater resistance to digestion by the protease. While the catalytic potential

of BLA was enhanced by proteolysis, that of BAA was diminished owing to this process.

Combined with greater catalytic activity, a lower thermal stability was observed for BLA on

proteolytic treatment. For both enzymes, the extent of proteolytic cleavage was reduced in

the presence of various stablizing agents.

The productivity of alpha-amylase is affected by various carbon and nitrogen sources.

Fukumoto et al. (1957) found that lactose and galactose were most effective in stimulating

amylase production. Glucose and fructose were most effective in promoting respiration but

were almost ineffective with regards to enzyme formation. However, glucose, which was

rather inhibitory at high concentration, was found to become available at low concentration.

Nomura et al. (1956) reported on the effect of several substrates on amylase formation.

Glucose and casein hydrolysate stimulate the alpha-amylase formation. The addition of

amino acid causes only a slight inhibition of enzyme formation.

The effects of different oilseed cakes on alpha-amylase production by

Bacillus licheniformis CUMC305 were investigated by T. Krishnan and A.K. Chandra

(1982). The oilseed cakes came from different sources namely groundnut, coconut copra,

sesame, madhuca, cotton mustard and linseed. All enhanced production of thermostable

extracellular alpha-amylase by Bacillus licheniformis CUMC305 but on varying degrees. The

effects of the different concentrations of each oilseed cake was compared to the results of a

control experiment in which Bacillus licheniformis was grown in conventional medium

without any oilseed cake. The saccharolytic alpha-amylase activity in the control experiment

was 6 U/ml, which was considered as 100%, and the relative enhancement of enzyme

production was calculated accordingly. The best effect was obtained with mustard seed cake,

10
which caused an increase in enzyme production of almost two-fold at concentration above

2%. Cottonseed cakes showed the next best effect as determined by direct correlation of the

concentration gradient with the enzyme at 1.75 fold increase.

It was evident from the study that oilseed cakes may serve as ideal fermentation bases

for obtaining high yields of alpha-amylase from Bacillus licheniformis CUMC305.

The existence of any relationship between extracellular alpha-amylase production and

sporulation in Bacillus licheniformis CUMC305 when grown in the presence of different

carbohydrates was investigated. It was noted that alpha-amylase production in the organism

was almost complete during the period of maximum sporulation, irrespective of the carbon

source used (Krishnan and Chandra, 1983).

Votruba et al., (1984) have studied the kinetics of alpha-amylase production in

continuous cultivation of Bacillus licheniformis on a semi-synthetic medium (glucose or

maltose as C source). The specific rate of alpha-amylase production was proportional to

growth rate but it was repressed by higher substrate concentrations. Besides glucose or

maltose, peptone was also used as an alternative carbon source during cultivation. The

specific rate of production of the enzyme on maltose was half that found with glucose.

Pratima and Umender (1989) studied the production of amylase in a low cost medium

by Bacillus licheniformis TCRD-B-13 that was isolated from soil. The alpha-amylase of this

strain showed excellent stability at high temperature and over a wide pH range. Elimination

of yeast extract and peptone from the basal cornstarch medium and substitution by gluten

resulted in higher enzyme concentration in the fermented broth. The addition of corn flour to

the medium further improved alpha-amylase production.

11
 Ramesh and Lonsane (1990) have studied the critical importance of moisture content

of the medium in alpha-amylase production by Bacillus licheniformis M27 in a solid-state

fermentation system. A large reduction (about 30-78%) was observed in the production of

alpha-amylase by Bacillus licheniformis M27 in standardized wheat bran medium under

solid-state fermentation when the moisture content of the medium was higher than the

standardized value (65%). However, a surge in enzyme production in the first 24 h of

fermentation was observed in media with 75% and 85% moisture. The role of decreased

oxygen transfer in reducing enzyme titers by about 78% in the medium containing 95%

moisture was evident, since the enzyme titer can be effectively increased by agitating the

medium during fermentation.

Ramesh and Lonsane (1991) found that amylase production by Bacillus licheniformis

M27 in submerged fermentation was reduced from 480 to 30 U/ml when soluble starch

concentration in medium was increased from 0.2 to 1.0%. In contrast the enzyme production

increased with 29-fold increase in the concentration of soluble starch and other starch

substrates in solid-state fermentation systems.

From the investigation of some properties of the alpha-amylase and proteinase in the

culture filtrate from Bacillus licheniformis MB 80 strain it has been established that the

alpha-amylase activity is the highest at pH 6.0 to 6.5 and at 90°C (Emanuilova et al., 1984).

Leaching of alpha-amylase from bacterial bran, produced by Bacillus licheniformis

M27 in solid-state fermentation was about 2.2 times higher at 50°C as compared to that at

30°C. Further increase by about 19% in leaching efficiency was observed when contact time

was extended from 60 to 120 min. The overall increase of 2.54 times under these strategies is

12
of economic importance and no information was available earlier on enhanced leaching of

enzyme from fermented bran at elevated temperatures (Padmanabhan et al., 1992).

Ivanova et al. (1993) purified the extracellular alpha-amylase by the Bacillus

licheniformis strain. The enzyme was stable at pH 6.5-8.0, while its optimum temperature

was 90°C. The thermostability was Ca2+ dependent. The half-life of the purified enzyme was

10 min at 85°C in buffer without Ca2+. The half-life at pH 6.5 with 1.0 mM CaCl 2 added was

30 min., and over 120 min. with 5.0 mM CaCl 2. the purified enzyme was strongly inhibited

by N-bromosuccinimide (NBS) and by EDTA.

Anyangwa et al. (1993) studied the effect of removal of starch in the sugar refining

industry by alpha-amylase from the bacterium Bacillus licheniformis. The optimum condition

for use of the enzyme was at temperature 80-95°C, pH 5.0-7.0 and the reaction time 30

minutes. The activity was increased with concentration of the enzyme.

The influence of polyhydric alcohols and carbohydrates on the thermostability and

the heat inactivation kinetics of Bacillus licheniformis alpha-amylase was studied in the

temperature range 96°C to 130°C. High concentrations (from 9 to 60 weight percent) of

glycerol, sorbitol, mannitol, sucrose, or starch can markedly decrease the inactivation rate

constant, k, and in the studied cases, this stabilizing effect grows stronger with increasing

additive concentration (Dc-Cordt et al., 1994).

Hendriekx et al. (1994) studied the influence of polyhydric alcohols and

carbohydrates on the thermostability of alpha-amylase. The heat inactivation kinetics of

Bacillus licheniformis alpha-amylase was studied in the temperature range 96°C to 130°C.

13
High concentrations of glycerol, mannitol, sucrose or starch can markedly decrease the

inactivation rate.

Weemaes et al., (1996) have compared three different alpha-amylases from Bacillus

subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis with respect to thermal

stability, pressure stability, and combined pressure-temperature stability. Measurements of

residual enzyme activity and residual denaturation enthalpy showed that the alpha-amylase

from Bacillus licheniformis has by far the highest thermostability and that two other alpha-

amylases have thermostabilities of the same order magnitude. FTIR spectroscopy showed

that changes in the conformation of the alpha-amylases from Bacillus amyloliquefaciens,

Bacillus subtilis, Bacillus licheniformis due to pressure occurred at about 6.5, 7.5, and 11

kilobar, respectively. It seemed that for the enzymes studied, thermal stability was correlated

with pressure stability. As to the resistance under combined heat and high pressure, the

alpha-amylase from Bacillus licheniformis was much more stable than the alpha-amylases

from Bacillus amyloliquefaciens and Bacillus subtilis, the latter two being about equally

stable. It appears that under high pressure or temperature, Bacillus licheniformis alpha-

amylase was most resistant among the three enzymes studied.

A study on the selection of a suitable low cost fermentation medium for the

production of alpha-amylase by Bacillus licheniformis GCBU-8 was conducted by Ikram-ul-

Haq et al. (2002). It utilized different agricultural by products such as wheat bran, sunflower

meal, cotton seed meal, soybean meal, and rice husk or rice bran. Wheat bran was found to

be the best basal and standardized medium for optimal production of alpha-amylase. The

production was increased 2-fold when soluble starch was replaced with pearl millet starch at

1% level and nutrient broth concentration was reduced from 1% level to 0.5%. The newly

14
selected fermentation medium contained (%w/v) wheat bran 1.25, nutrienth broth 0.5, pearl

millet starch 1.0, lactose 0.5, NaCl 0.5, CaCl2 0.2 in 100 ml of phosphate buffer. The

production of the enzyme was greater in the newly selected medium compared to the

conventional medium.

A follow-up study was conducted by Ikram-ul-Haq et al. (2005) on starchy substrates

for the production of alpha-amylase by parental and mutant derivatives of Bacillus

licheniformis. The starches were added to the fermentation medium at 1% level. The

production of enzyme by the parental strain was higher in the presence of soluble starch.

However, its mutant derivatives gave optimum production of alpha-amylase in the presence

of pearl millet starch. This was attributed to the adequate nutrient content of pearl millet

(carbohydrates 67.1%, protein 11.6%, minerals 2.7%) for the growth of microorganism as

well as for the production of alpha-amylase. The parental and its mutant derivatives were

compared for the production of alpha-amylase in the presence of pearl millet. The pearl

millet was added to the fermentation medium at 0.5-3.0% levels. Maximum enzyme activity

by mutant strain GCBU-8 was achieved when 1.0% starch was added to the medium, while

in the other mutant derivatives optimum alpha-amylase production was when 1.5% pearl

millet was supplemented in the medium. As the amount of the starch was further increased,

the growth of the organism and the enzyme production were significantly inhibited. The

effect of different concentrations of nutrient broth was also investigated. The nutrient broth

was added to the medium at 0.25-1.25% levels. The parental strain gave maximum enzyme

production in the presence of 1.0% nutrient broth and 0.5% in the mutant derivatives, but for

Bacillus licheniformis GCUCM-30 it was 0.25% of nutrient broth. As the amount of the

nutrient broth was increased, the productivity was significantly decreased, possibly due to the

15
fact that the higher concentration of nitrogen source has an adverse effect on the growth of

microorganisms as well as on the production of alpha-amylase (Hewitt and Solomos, 1996;

Pedreson and Nielson, 2000).

P.V. Dharani Aiyer (2004) studied the effect of C:N ratio on alpha-amylase

production by Bacillus licheniformis SPT 27, an isolate obtained from the soil of Cambay, in

the western region of Gujarat, India. It produces extra cellular alpha-amylase exhibiting

activity at a wide pH range and was relatively stable. The Bacillus licheniformis isolate,

however, produces low yields of the amylase. Different types of starchy grains and tubers

were tested on its effect on the amylase activity. Amaranthus paniculatus has the highest

activity followed by Zea mays, potato, and Metroxylan remphii. Of the nitrogen sources

tested, peptone and ammonium hydrogen phosphate were best organic and inorganic sources,

respectively. The C: N ratio found to be optimum was 1:1.

A study conducted by David M. Rothstein et al. (1986) showed that alpha-amylase

production varied more than 100-fold depending on the presence or absence of a catabolite-

repressing carbon source in the growth medium with Bacillus licheniformis as the

microorganism. Alpha-amylase production by Bacillus licheniformis is related to the nature

of carbon source used for growth. If glucose is present in ample amounts, alpha-amylase is

repressed, even if starch is present. When glucose is the carbon source, but present in growth-

rate-limiting concentrations, then alpha-amylase production increases. The alpha-amylase in

Bacillus licheniformis 5A1 was produced predominantly during the growth phase and not at

the onset of the stationary phase. It was also shown in the study that transcription was

enhanced at least 50-fold when glutamate was used as carbon source, consistent with the

observed increase in secreted alpha-amylase. Cells utilizing glutamate as a carbon source

16
initiate a considerable number of alpha-amylase transcripts consistent with strong expression

of the alpha-amylase gene. Regulation of alpha-amylase clearly occurs at the level of

transcription.

In Central Amazonia, Brazil a study was conducted using six isolates of indigenous

rhizobia, which were screened for the production of amylases in liquid media using various

starchy substances as carbon sources (Oliveira A. N. et al., 2006). The effects of the wheat

bran, cassava, oat, peach palm, potato, tapioca flour, corn starch, and maltose on growth and

amylase production were studied after adding 1.0% (w/v) substrates to the modified YM

medium. All rhizobia strains could produce more extracellular protein, biomass and amylases

with the different kinds of carbon substrates. Among the carbon sources tested, maltose was

the best substrate for protein and amylase production. In general, peach, palm flour and

cornstarch were also considered to be good carbon sources for rhizobia amylases. The

biomass production by the rhizobia isolates was higher in the presence of oat flour. The

results obtained in the study revealed several Central Amazonian rhizobia strains as

promising sources of amylase for biotechnological applications especially in starch industry.

Saito and Yamamoto (1975) investigated the effects of various carbohydrates on

alpha-amylase synthesis. Polysaccharides such as glycogen, starch, and dextrin induced

alpha-amylase formation when such polysaccharides were added to a culture pre-grown on

sorbital medium. Induction of alpha-amylase also resulted from addition of maltose

oligosaccharides, maltotriose, maltotetraose, maltopentose, maltohexaose, and

maltoheptaose. All have been found to be superior to maltose as inducers of alpha-amylase

synthesis in Bacillus licheniformis 584. Amylase synthesis in the parent strain resulted by the

17
addition of compounds having linkages of α -1,4-, β -1,4-, and β -1,6- glucosyl glucose, or

α -1,6- galactosyl glucose.

Studies conducted by D. Dhanasekaran et al. (2006) compared the production of

alpha-amylase using Bacillus species on free and immobilized state. It has utilized Bacillus

although the particular specie was not specified. Cells of Bacillus species were immobilized

by entrapment in sodium alginate for the production of alpha-amylase. Cell growth rate was

reduced when the cells were immobilized as compared to free cells. Reduced growth rate of

immobilized cells may be attributed to the mass transfer limitation of oxygen. Maximum

concentration of alpha-amylase was obtained at 48 hours by the isolates. Immobilized cells

did not show any appreciable alpha-amylase production. The alpha-amylase of Bacillus sp.

had the optimum pH at 6.5-7.5 and temperature optima at 45°C, with the maximum activity

in substrate concentration of 0.1% to 1.0%. The characterization of the type of amylase was

done using thin layer chromatography.

Bacteria can only grow and multiply in the presence of a source of energy, which can

be derived from the controlled breakdown of various organic substrates present in the

external environment such as polysaccharides, lipids, and proteins. It is a known fact that

some members of genus Bacillus are able to breakdown starch and utilize it as source of

energy. Organisms unable to produce amylase cannot utilize starch as energy. Students of

Emphoria State University, Emporia, Kansas (USA) examined the ability of various Bacillus

species to regulate expression of the enzyme amylase (D. M. Nickless et al., 2001). This was

done by growing the organisms in the presence of different sugars and amylase activity was

assayed on a starch plate.

18
 Alpha-amylase levels in Bacillus licheniformis 5A1 were compared in culture grown

in medium containing either glucose or starch as a sole carbon source. Cells were harvested

during exponential growth and in stationary phase. Approximately five times as much alpha-

amylase was present per cell mass when cells were grown in medium containing starch

instead of glucose. For a given carbon source alpha-amylase synthesis remained constant

during exponential growth. In the study of D. Rothstein et al. (1986) it was shown that alpha-

amylase production in Bacillus licheniformis is related to the nature of carbon source used for

growth. If glucose is present in ample amounts alpha-amylase is repressed, even if starch or

other carbon source are also present. The extent to which catabolite repression is relieved

correlates with the degree of growth limitation of the carbon source. Growth medium

containing starch is not a particularly depressing condition for alpha-amylase. Bacillus

licheniformis 5A1 is regulated differently from most Bacilli. Strain 5A1 and other Bacillus

licheniformis strain grow well in defined medium unlike other Bacilli that require undefined

supplements that could influence alpha-amylase production. It was also showed that growth

conditions resulting in the appearance of alpha-amylase in the culture medium result in a

corresponding increase in transcription from the alpha-amylase promoter. Regulation of

alpha-amylase occurs at the level of transcription. The data confirmed the presence of a

transcription termination site upstream of the alpha-amylase promoter at –6S at a location

following inverted repeats and a poly (T) tract, typical of prokaryotic termination sites.

The appearance of alpha-amylase in the culture medium of Bacillus licheniformis 584

has the characteristics of de novo protein synthesis during the period of secretion and

controlled by induction, catabolite repression, apparent temperature-sensitive repression, and

culture age (Saito and Yamamoto, 1974). Amylase formation in the parent strain of Bacillus

19
licheniformis was immediately suppressed by the addition of 0.5% glucose, glycerol, acetate

or succinate, and no measurable enzyme synthesis occurred when glucose or starch were

added simultaneously to non-induced cultures of the parent strain. These observations

suggest that the secretion of alpha-amylase in the parent strain is sensitive to catabolite

repression. The addition of cyclic AMP to a growing culture of the parent strain at various

points stimulated the enzyme production by about 40-70% but could neither shorter the lag

period after which the enzyme synthesis starts nor result in an alleviation of the repressive

effect by glucose. This observation did not occur in other mutant strains in which alpha-

amylase formation was not repressed by 0.5% glucose but was sensitive to 2% glucose. It

was observed that mutants F-12 and F14 could synthesize alpha-amylase when grown on a

medium containing glucose as the sole carbon source.

The age of the culture fluid is important factor in promoting alpha-amylase synthesis

by Bacillus licheniformis (Saito and Yamamoto, 1974). In the study, it showed that the best

combination for alpha-amylase formation was two-day-old cells and one day-old culture

fluid. In this case, however, alpha-amylase formation was increased by the addition of either

0.5% starch or glucose and no catabolite repression was observed at this concentration.

20
 Chapter 3

ALPHA-AMYLASE PRODUCTION BY BACILLUS LICHENIFORMIS 1331 USING

JACKFRUIT SEEDS AS SUBSTRATE

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

Introduction

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 used as a substrate for alpha-amylase production

21
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 can 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 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

22
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.

Methodology

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.

Bacterial strain

23
 The bacteria, Bacillus licheniformis 1331, was obtained from the Philippine National

Collection of Microorganisms (PNCM), BIOTECH, University of the Philippines, Los

Baños, Laguna.

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.

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 incubated at 30°C

for 18 h.

Production medium

24
 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.

Optimization of cultural conditions

Factors such as temperature and source of carbon and nitrogen affecting production of

amylase were optimized by varying parameter one at 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)

Nitrogen source (w/v) 10% 15% 20%

20% (10%, 20%) (15%, 20%) (20%, 20%)

Table 2. Factorial design at pH 7 and 37°C

Carbon source (w/v)

Nitrogen source (w/v)

10% 15% 20%
20% (10%, 20%) (15%, 20%) (20%, 20%)

25
 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.

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).

Alpha (α )-amylase assay

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.

Buffer solution

The KH2PO4 solution was a mixture of 0.6803 g of KH 2PO4 (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.

Substrate: Starch solution

26
 0.2 g soluble starch was dissolved in boiling 0.05M KH 2PO4-NaOH buffer at pH 6.0

and was cooled to 40°C.

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:

Ac − At
α -Amylase activity (U/ml)= × 40 D , where: Ac is the absorbance of
Ac

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).

Protein determination by Bradford assay

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

27
hour, after which the absorbance of each solution was measured at 595 nm, using distilled

water as a blank.

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.

If the enzyme solution has absorbance reading outside the range 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.

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.

28
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)

10% Carbon, 20% Nitrogen 0.1592
15% Carbon, 20% Nitrogen 0.059
20% Carbon, 20% Nitrogen 0.1061

29
 0.18

Alpha-amylase activty (U/ml)

0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
1 2 3
% Carbon, % Nitrogen

Figure 1. Alpha-amylase activity at pH 7 and 30°C

The average of the results of the alpha-amylase activity for three trials at pH 7 and

37°C is shown in Table 4. The highest alpha-amylase activity was also at 10% carbon and

20% nitrogen.

Table 4. Alpha-amylase assay at pH 7 and 37˚C

Alpha-amylase activity (U/ml)

10% Carbon, 20% Nitrogen 0.6374
15% Carbon, 20% Nitrogen 0.609
20% Carbon, 20% Nitrogen 0.1658

30
 0.7

Alpha-amylase activity (U/ml)

0.6

0.5

0.4

0.3

0.2

0.1

0
1 2 3
% Carbon, % Nitrogen

Figure 2. Alpha-amylase activity at pH 7 and 37°C

Figure 1 and Figure 2 shows that the higher concentration of carbon source has an

adverse effect on the production of alpha-amylase particularly at 37°C. It may be due to the

thickness of the fermentation medium that resulted in the decreased agitation and poor

mixing of air, which were essential for the growth of organism and production of enzyme

(Haq et. al., 1998). Also, alpha-amylase production was possibly affected by the presence of

a catabolite repressing carbon source in the growth medium.

Table 5. Protein concentration at pH 7 and 30°C

Protein concentration (mg/ml)

10% Carbon, 20% Nitrogen 3.1338
15% Carbon, 20% Nitrogen 3.3168
20% Carbon, 20% Nitrogen 3.1464

31
 3.35

Protein Concentration (mg/ml)

3.3

3.25

3.2

3.15

3.1

3.05

3
1 2 3
% Carbon, % Nitrogen

Figure 3. Protein concentration at pH 7 and 30°C

Table 6. Protein concentration at pH 7 and 37°C

Protein concentration (mg/ml)

10% Carbon, 20% Nitrogen 2.8833
15% Carbon, 20% Nitrogen 2.7915
20% Carbon, 20% Nitrogen 3.0993

32
 3.15

Protein Concentration (mg/ml)

3.1
3.05
3
2.95
2.9
2.85
2.8
2.75
2.7
2.65
2.6
1 2 3
% Carbon, % Nitrogen

Figure 4. Protein concentration at pH 7 and 37°C

The total protein concentration in the sample was determined by the Bradford assay.

As shown in Table 5 and Table 6, the highest protein concentration was obtained at 30°C,

15% carbon and 20% nitrogen.

Table 7. Alpha-amylase activity, Protein concentration, and Biomass concentration of

samples taken at regular intervals
Time (h) Alpha-amylase activity (U/ml) Protein concentration (mg/ml) Biomass (mg/ml)
0 0.2879 2.6988 0.2
1 0.3165 2.9745 0.26
2 0.3275 3.0455 0.2
4 0.4045 3.2888 0.36
6 0.5868 3.3288 0.38

33
 8 0.6484 3.224 0.3
18 0.6593 3.2825 0.22
24 0.6593 3.2975 0.32
27 0.6659 3.2318 0.12
30 0.6725 3.2688 0.38

3.5
Concentrations (mg/ml)

2.5 Alpha-amylase activity

(U/ml)
2 Protein concentration
1.5 (mg/ml)
Biomass concentration
1 (mg/ml)

0.5

0
0 10 20 30 40
time (h)

Figure 5. Plot of Alpha-amylase activity, Protein Concentration, and Biomass Concentration

during course of fermentation
The production of alpha-amylase was conducted in 500 ml of medium at pH 7 and

optimum temperature of 37°C and C/N ratio of 10% carbon and 20% nitrogen. Samples were

taken at regular intervals (0, 1, 2, 4, 6, 8, 18, 24, 27, and 30h) for analysis of alpha-amylase

activity, protein concentration and biomass (DCW), as shown in Table 7.

Figure 5 shows the alpha-amylase activity in the course of fermentation as shown in

Alpha-amylase activity was highest during exponential growth at 4-8 hours and protein

concentration was highest at 6 hours of fermentation.

The values of protein concentration in mg/ml were higher than the values of alpha-

amylase in U/ml. It may be due to the fact that protein concentration in jackfruit seeds was

high.

34
 Ajayi (2008) reported that the moisture, ash, protein, crude oil, crude fiber, and

carbohydrate contents of Artocarpus heterophyllus seeds to be 2.78%, 6.72%, 20.19%,

11.39%, 7.10%, and 51.82%, respectively. Bobbio et al., (1978) reported protein, crude

lipids, and carbohydrate contents of jackfruit seeds as 31.9%, 1.3%, and 66.2%, respectively.

Kumar et al. (1988) also reported composition of seeds from two varieties of jackfruit.

Protein, crude lipids, and carbohydrate content were 17.8-18.3%, 2.1-2.5%, and 76.1%,

respectively. The protein content of Artocarpus heterophyllus reported by Bobbio et al.

(1978) was very high; however the seeds were reported to have been collected from fruits of

various varieties of jackfruit.

Table 8. Yields and Productivity

Time (h) Yamylase/protein Yp/x Productivity

0 0.1067 1.4395 N/A
1 0.1064 1.2173 0.3165
2 0.1075 1.6375 0.1637
4 0.123 1.1236 0.1011
6 0.1763 1.5442 0.0978
8 0.2011 2.1613 0.081
18 0.2009 2.9968 0.0366
24 0.2 2.0603 0.0274
27 0.2061 5.5492 0.0247

35
 30 0.2057 1.7697 0.0224

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.

36
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.

Recommendation

In this study, the production medium contained the basic ingredients that would make

the bacteria produce alpha-amylase enzyme. Determining the best carbon source and nitrogen

source could improve alpha-amylase production by Bacillus licheniformis 1331.

In the study, 10% carbon and 20% nitrogen (1:2) was found as the best C/N ratio. It is

recommended that other less expensive nitrogen sources such as corn steep liquor, peptone,

and ammonium hydrogen phosphate be used.

It is also recommended that other investigators do following study on thermal

stability, characterization of alpha-amylase by using SDS-PAGE and zymogram, as were as

the production and purification of enzyme in large scale.

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.

Arunava, B., Pal, S.C. and S.K. Sen (1993). Alpha-amylase production in lactose medium by
Bacillus circulanse. J. Microbiologia., Volume 9 (2),142-148.

37
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.

38
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

39
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.

40
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.

41
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.

42
 Chapter 4

CONCLUSION

Alpha amylase was successfully produced using jackfruit seed as 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 (10% carbon and 20%

nitrogen). Alpha-amylase activity was highest during exponential growth at 4-8 hours of

fermentation. Protein concentration was highest at 6 hours of fermentation.

43
 Chapter 5

RECOMMENDATION

In this study, the production medium contained the basic ingredients that would make

the bacteria produce alpha-amylase enzyme. Determining the best carbon source and nitrogen

source could improve alpha-amylase production by Bacillus licheniformis 1331.

In the study, 10% carbon and 20% nitrogen (1:2) was found as the best C/N ratio. It is

recommended that the other less expensive nitrogen sources such as corn steep liquor,

peptone, and ammonium hydrogen phosphate be used.

It is also recommended that other investigators do follow-up study on thermal

stability, characterization of alpha-amylase by using SDS-PAGE and zymogram, as well as

production and purification of enzyme in large scale.

44
 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.

Arunava, B., Pal, S.C. and S.K. Sen (1993). Alpha-amylase production in lactose medium by
Bacillus circulanse. J. Microbiologia., Volume 9 (2),142-148.

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.

45
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.

46
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

47
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.

48
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.

49
APPENDICES
 APPENDIX A
Composition of HIMEDIA M001

Table A.1 Composition of HIMEDIA M001

Standard Formula

Ingredients Concentrations (g/L)

Peptic digest of animal tissue 5
Beef extract 1.5
Yeast extract 1.5
Sodium Chloride 5
Agar 15

Final pH (at 25°C) 7.4 ± 0.2

 APPENDIX B
Assay on Alpha-amylase Activity

Table B.1 Alpha-amylase activity at pH 7 and 30°C

Alpha-amylase activity Average

Absorbance (U/ml) (U/ml)
Substrate Control 0.0157
10% Carbon, 20% Nitrogen Trial 1 0.0174 -0.10828054
Trial 2 0.0126 0.197452229 0.1592
Trial 3 0.0138 0.121019108

15% Carbon, 20% Nitrogen Trial 1 0.0147 0.063694267

Trial 2 0.0145 0.076433121 0.059
Trial 3 0.0151 0.03821656

20% Crabon, 20% Nitrogen Trial 1 0.0139 0.114649681

Trial 2 0.0147 0.063694267 0.1061
Trial 3 0.0135 0.140127388

Table B.2 Alpha-amylase activity at pH 7 and 37°C

Alpha-amylase activity Average

Absorbance (U/ml) (U/ml)
Substrate Control 0.0434
10% Carbon, 20% Nitrogen Trial 1 0.0183 0.57831013
Trial 2 0.0155 0.642857142 0.6374
Trial 3 0.0134 0.691244239

15% Carbon, 20% Nitrogen Trial 1 0.0243 0.440092165

Trial 2 0.0151 0.652073732 0.609
Trial 3 0.0115 0.735023041

20% Carbon, 20% Nitrogen Trial 1 0.0401 0.076036866

Trial 2 0.0312 0.28110599 0.1658
Trial 3 0.0373 0.140552995

Computation:
Ac − At
α -Amylase activity (U/ml)= × 40 D
Ac
Ac= absorbance of substrate control
At= absorbance of test sample
40= 4.0 mg starch present in the reaction tube times 10
D= enzyme dilution factor
 APPENDIX C
Assay on Protein Concentration

BSA Standard Curve

Table C.1 Tabulation of absorbance versus BSA concentration

BSA concentration
(ug/mL) Absorbance
0 0
125 0.0414418
250 0.1344418
500 0.2535418
1000 0.4144418
1500 0.6321418

0.7

0.6
y = 0.0004x + 0.0123
Absorbance 595 nm

0.5 R2 = 0.9921

0.4

0.3

0.2

0.1

0
0 200 400 600 800 1000 1200 1400 1600
BSA Concentration, ug/mL

Figure C.1 Plot of BSA standard curve

Table C.2 Protein concentration at pH 7 and 30°C
Dilution factor: 0

Protein Concentration Average

Trial Absorbance (g/L) (mg/ml)
10% Carbon, 20%
Nitrogen 1 1.2459 3084
2 1.308 3239.25 3.1338
3 1.2436 3078.25

15% Carbon, 20%

Nitrogen 1 1.3387 3316
2 1.2407 3071 3.3168
3 1.4376 3563.25

20% Carbon, 20%

Nitrogen 1 1.2687 3141
2 1.2767 3161 3.1464
3 1.2672 3137.25

Table C.3 Protein concentration at pH 7 and 37°C

Dilution factor: 0

Protein Concentration Average

Trial Absorbance (g/L) (mg/ml)
10% Carbon, 20%
Nitrogen 1 1.1669 2886.5
2 1.2175 3013 2.8833
3 1.1125 2750.5

15% Carbon, 20%

Nitrogen 1 1.0205 2520.5
2 1.1039 2729 2.7915
3 1.2623 3125

20% Carbon, 20%

Nitrogen 1 1.3225 3275.5
2 1.3114 3247.75 3.0993
3 1.1222 2774.75
 APPENDIX D
Alpha-amylase activity, Protein Concentration, and Biomass Concentration of
Samples Taken at Regular Intervals

Table D.1 Alpha-amylase activity of samples taken at regular intervals

Alpha-amylase activity
Absorbance (U/ml)
Substrate Control 0.0455
Time (hour)
0 0.0324 0.287912087
1 0.0311 0.316483516
2 0.0306 0.327472527
4 0.0271 0.404395604
6 0.0188 0.586813186
8 0.016 0.648351648
18 0.0155 0.659340659
24 0.0155 0.659340659
27 0.0152 0.665934065
30 0.0149 0.672527472
Alpha-amylase activity (U/ml)

0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0 5 10 15 20 25 30 35
Time (h)

Figure D.1 Plot of Alpha-amylase activity of samples taken at regular intervals

Table D.2 Protein concentration of samples taken at regular intervals

Time Concentration Concentration

(hour) Absorbance (g/L) (mg/ml)
0 1.0918 2698.75 2.6988
1 1.2021 2974.5 2.9745
2 1.2305 3045.5 3.0455
4 1.3278 3288.75 3.2888
6 1.3438 3328.75 3.3288
8 1.3019 3224 3.224
18 1.3253 3282.5 3.2825
24 1.3312 3297.25 3.2973
27 1.305 3231.75 3.2318
30 1.3198 3268.75 3.2688
Protein concentration (mg/ml)

3.5
3
2.5
2
1.5
1
0.5
0
0 5 10 15 20 25 30 35
Time (h)

Figure D.2 Plot of Protein concentration of samples taken at regular intervals

 Table D.3 Biomass concentration of samples taken at regular intervals

Preweighted micro test tube Dried sample in micro test tube Dry weight of biomass
Time (h) (g) (g) (g)
0 7.5 8.5 1
1 7.5 8.8 1.3
2 7.6 8.6 1
4 8.7 10.5 1.8
6 7.5 9.4 1.9
8 7.7 9.2 1.5
18 5.4 6.5 1.1
24 5.3 6.9 1.6
27 7.8 8.4 0.6
30 8.7 10.6 1.9

Dry weight of biomass Biomass concentration

(g/L) (mg/ml)
200 0.2
260 0.26
200 0.2
360 0.36
380 0.38
300 0.3
220 0.22
320 0.32
120 0.12
380 0.38
 Biomass concentration (mg/ml)
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0 5 10 15 20 25 30 35
Time (h)

Figure D.3 Plot of Biomass concentration of samples taken at regular intervals

Table D.4 Specific enzyme activity (Yamylase/protein), Yp/x, and Productivity of samples taken
at regular intervals

Time Alpha-amylase activity Protein Concentration Biomass

(h) (U/ml) (mg/ml) (mg/ml)
0 0.287912087 2.69875 0.2
1 0.316483516 2.9745 0.26
2 0.327472527 3.0455 0.2
4 0.404395604 3.28875 0.36
6 0.586813186 3.32875 0.38
8 0.648351648 3.224 0.3
18 0.659340659 3.2825 0.22
24 0.659340659 3.29725 0.32
27 0.665934065 3.23175 0.12
30 0.672527472 3.26875 0.38

Specific enzyme Productivit

activity Yp/x y
0.106683497 1.439560435
0.106398896 1.217244292 0.316483516
0.107526688 1.637362635 0.163736264
0.122963316 1.123321122 0.101098901
0.17628635 1.544245226 0.097802198
0.201101628 2.16117216 0.081043956
0.200865395 2.997002995 0.036630037
0.199966839 2.060439559 0.027472527
0.206059895 5.549450542 0.024664225
0.205744542 1.769809137 0.022417582