UNIVERSITI PUTRA MALAYSIA
PRODUCTION AND CHARACTERIZATION OF THERMOSTABLE AMYLASES FROM BACILLUS CIRCULANS ISOLATED
FROM A LOCAL HOT SPRING
SHARIFAH SHAHRUL RADIAH DT. SYED ALWEE
FSMB 1992 3
PRODUCTION AND CHARACTERIZATION OF THERMOSTABLE AMYLASES FROM BACILLUS CIRCULANS ISOLATED
FROM A LOCAL HOT SPRING
By
SHARIFAH SHAHRUL RADIAH DT. SYED ALWEE
Thesis Submitted i n Ful f i lment o f the Requirements for the Degree of Mas te r of Science in the Faculty of
Food Science and Biotechnology, Universiti Pertanian Malaysia
Sep tembe r 1992
ACINOWLEDGEttENTS
The author wishes to express her sincere appreciation to
the following:
Dr. Baharuddin Abdul Ghani and Dr. Hohd. Ismail Abdul
Karim for their guidance, support and encouragement throughout
the author's graduate studies and in the preparation of this
thesis.
Hr. Hohd. Khalid Sikas, Hiss Rosmah Jaafar, and Puan siti
Junaidah for their technical assistance and advice.
Hr. Abdul lIadi for typing this thesis.
Finally, the author would like to thank her parents for
being so patient throughout her graduate studies.
ii
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS. . . . • • • • • • . . . . • . • • • • • • • . • • • • • • • • • • • • • • • i i
LIST OF TABLBS......................................... vii
LIST OF FIGURES........................................ vii i
LIST OF PLATES......................................... x
ABSTRACT. . . . . . . . . . . . . . • • . . . . . . . . • • . . • • . • . • • • • • . • • . . . . . • xi
ABSTRAK.. . . . . . . . . . . . . • . • . • . . . . . . • • • . . . • . • • • • . . . . . . . . . • • xiii
CHAPTER
I
II
INTlODU<:'I'ION' • • . • . . • . • • e, • • • • • • • • • • • • • • • • • • • • • 1
LITERATURE REVIEW . . . . • . . . . . . . . . . . . . . . . . . • . . . 5
Starch: The Substrate . . . . . . • • • • • . . . • • • • • . 5
Production of a-Amylase . . . . . . . . . . . • • . . . . • • 10
Amylase-Producing Bacteria • . • . • • • • . • . . • 10
Effects of Environmental Factors on a-Amylase Production . . . . . • • . • . • . . . • . . . • 13
Genetic Regulations to Increase a-Amylase Production . . • . . . • • . . . • . . . . • • • 17
Hode of Action of a-Amyl ase • • • • • • • • • • • • • • • 20
Properties of a-Amylase . . . . • . • • . . . • . • . . . . . 26
Molecular Weight of a-Amylase . • . • • . • . . . 26
Thermal Stability of a-Amylase . • • • • • • . . 27
Effects of pH on Stability of a-Amylase 31
Effects of Metal Ions on Stability and Activity of a-Amylase . . . • • • • • • • • • • • 32
iii
Page
Purification and Homogeneity of Purrified a-Amylase • • • • . . . • . . . . . • . . • • • • 34
Regulation of Thermostable a-Amylase in Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Catabolite Repression • . . • . . • . . • . • . • . . . . 36
Substrate Induction . . . . . . . . . . . • . . • • . . . • 39
Applications of Thermostable a-Amylase 41
III MATERIALS AND METHODS
Equipments . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 45
Chemicals . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . 47
Reagen ts • • • . . • . • • • . . • • . • • • • • • • • • • . • • • • • 47
Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Isolation of Amylolytic Bacteria 52
Screening and Purification of Amylolytic Bacteria • • . . . . . . . • • • • . . • • • • • 53
Maintenance of Amylolytic.Bacteria . • • . . 53
Identification of Isolates . • • • • . . . • • • . . 53
CuI ture Conditions . . . . . . • • • • • • • • • . • • • • • • • • 55
Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 55
Assay of Amylase Activity ............. . 55
Reducing Sugar Content . • . . • • • . . . . . . . . . • 56
Protein Content Determination • . • . . . . • . • 57
Determination of Growth . . . . . . . . . . . • . . • . 57
iv
Page
Production of Amylase • • . • • • • • • • • • • • • • . • • • • 51
Effect of Time on Amylase Production 51
Effect of Temperature and pH on Amylase Production • • • • . • • • • • • • • • • • . • • . • 58
Effect of Carbon Sources on Amylase Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Purification of Amylase • • . • • • • • • • . • • . • • • • • 59
Determination of Molecular Weight by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis • • • • • • • • • • • • • • • . • • • . • . • 60
Properties of Purified Amylase • • • • • • • • • • • 61
pH Activity Profile 61
Temperature Activity Profile • . • • • • • . . • • 62
Effect of pH on Amylase Stability . . . . . . 62
Thermal Stability of Amylase . . • . . • • • • . • 62
Substrate Preference of Amylase . . • • . . • • 62
Thin-layer Chromatography of Hydrolysis Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 63
IV RESULTS AND DISCUSSIONS
Isolation, Screening and Identification of Amylolytic Bacteria • . • • • • • • • • • • • • • • • • • • 65
Growth Characteristics of Bacillus circulans strains SB-l and SB-2l • . . • . • • • . • 10
Amylase Production Profile of Bacillus circulans strains SB-l and SB-2l • . . . . . • . • . 13
Effect of Time on Amylase Production 7 3
Effect of Temperature and pH on Amylase Production . . . . • . . . • . . . • . . • • . • • • 75
v
v
Page
Effect of Carbon Sources on Amylase Production .. . . . . .. . .. . . ...... . ... . .. . .. 77
Purification of Bacillus circulans strain SB-l Amylase . . . . . . . . . . . . �................. 85
Characterization of the Purified Bacillus circulans Strain SB-l Amylase . . • • • . • • • • • • • 81
pH and Temperature Activity Profile of Purified Amylase . . • • . . . • • • • . • • • • . • . • • . • 81
pH and Thermal Stability of Purified Amylase . . .. .. . .. .. .. ...... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . ...... 89
Substrate Specificity of Purified Amylase .. .. .. .. .. .. .. . -. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 94
Thin-layer Chromatography Profile of Hydrolysis Products of Purified Amylase 98
CONCLUSION ........... .................................................. .. 105
BIBLIOGRAPHY .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 108
APPENDIX .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 116
BIOGRAPHICAL SKETCH . . • • • • • . . . • . • • • • . • • • • . • • . • • • . . . • • • • 126
vi
LIST OF TABLES
Table
I Properties of Starch Granules • • • • • • • • • • • • • • • • •
2 Amylose and Amylopectin Content of Various
Page
6
Starches .. . . . '................................. 7
3 Preliminary Tests for the Identification of Gram Positive Bacteria • • • • • • . • • • • • • • • • • • • • • • • • 68
4
5
6
Results of Biochemical Tests for the Determination of Bacillus sp • • . • • • • • • • • • . • • • • •
Results from the API 50 CHB Test • • • . • • • • • • • • • .
Parameters Observed During the Purification of Bacillus strain SB-l Amylase • • • . • • • . • • • • • • •
vii
70
71
89
Figure
1
2
3
LIST OF FIGURES
structure of Amylose
Structure of Amylopectin
62-a-maltosylmaltotriose
Page
7
9
4 Liquefaction and Saccharification of Starch
2 2
43
5 Characteristics for the Identification of Gram Positive Bacteria . . . • • • • • . . . . • • • . • • . • • • • • . 54
6 Growth Profile of Bacillus circulans strains 8B-l and 8B-21 . . • . . . • . . . . . • • • • • • . . . . . . • . • . . • • • . 72
7 Profile of Amylase synthesis by Bacillus circulans ...................................... 74
8 Effect of Temperature on Amylase Synthesis by Bacillus circulans strains SB-l and SB-21 . . . . • . 76
9 Effect of pH on Amylase Synthesis by Bacillus circulans strains SB-l and SB-2l . . . . • . . . . . . • • • . 78
10 Effect of Carbon Sources on Amylase Synthesis by Bacillus circulans . • . . . • . . . . • . . • • . . • . . . • . • • . 80
11 Effect of Glucose and Maltose on'
Amylase Synthesis by Bacillus circulans • . . . • • . . • • • • • • • . 82
12 Induction and Repression of Amylase Synthesis by Bacillus circulans . . . . . . . . . . . . . . . . . . . '.......... 84
13 First Purificatioll Profile of Bacillus circulans strain 8B-l Amylase . . • . . . . . . . . . . . . . • • . • • . . . . . • . 86
14 Second Purification Profile of Bacillus circulans strain SB-l Amylase . . . . . • . . . • . . . . . . • . 88
15 Determination of Molecular Weight of Purified Bacillus circulans strain 8B-l Amylase • . . . • . . • . 91
16 Temperature Activity Profile of Purified Bacillus circulans strain SB-l Amylase . • • . • . . • . 92
viii
17 pH Ac tivity Profile of Purified Bacillus circulans s t rain 8B-l Amylase . . . . . • • • . . . . . . • . . . 93
18 Thermal Stability of Purified Bacillus circulans strain SB-l Amylase . . . . . . . • • . . . . . . . . . . . . . . . . . . . 95
19 pH Stability -of Purified Bacillus circulans st rain SB-l Amylase . . . . . . . . . . . . . . . . . . . . . . • . . . . . 96
20 Subs trate Specifity of Purified Bacillus circulans s train 8B-l Amylase . . . . ..... . . . . . . . . . 97
21 Affinity of Purified Bacillus circulans s t rain SB�l Amyl ase Towards Mal todext rins . . . . . . . . . . . . . 99
ix
LIST OF PLATES
Plate
I Clearing Zone Formed by the Selected Colonies
2 Sodium Dodecyl Sulfate-Polyacrylamide Gel . • •
3 Thin-Layer Chromatography Plates showing Profile of Hydrolysis Products from Various
Page
67
90
Dextrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
4 Thin-layer Chromatography Plates Showing Profile of Hydrolysis Products from Various Starches . • • • . • • . • . . . . • • • • • • . • • • . • • • • 101
x
Abs tract of the s i s submi tted to the S enate of Universi t i Pertanian Malays i a as fulf i lment of the requirements f o r the degree of Master of Science .
Chairman
Faculty
PRODUCTION AND CHARACTERIZATION OF THERMOSTABLE AMYLASES FROM BACILLUS CIRCULANS ISOLATED
FROM A LOCAIJ 1I0T SPRING
By
SHARIFAH SRAHRUL RABIAH BTE SYED ALiEE
SEPTEMBER 1992
Dr. Baharuddin Abdul Ghani
Food Science and Biotechnology .
Two strains of amylol ytic Baci l lus were i solated from a
hot spring in Negeri Sembi lan and were ident i fi ed as Bac i l lus
ci rculans and desi gnated as stra ins 8B-l and SB-2 1 .
The optimal tempe rature and p H f o r g rowth a n d e n z y m e
production by both s trains were found t o b e at 5 5 ° C and pH
7 . 0 , respectively . The enzyme was produced f rom the beginning
of g rowth and reached max imum p r oduc t i on a t 7 2 hou r s . The
production of amylase was partially induced and production
occurred only in the presence of 1% s tarch . The presence of 20
mM m a l tose or mal tot r i os e enha n c e d e n z ym e p rodu c t i on . The
production was found to be repressed b y 20 mM glucos e .
The crude enzyme preparation of 88-1 was pur i f ied through
i on-exchange chromatography after 20- 4 0% ammon i um s u l f a t e
xi
precipitation and ultrafiltration. A single activity peak and
31 .6\ yield was obtained with a 53.9 fold purification . Using
SDS-PAGE the enzyme was shown to be homogenous and the
molecular weight of the purified amylase was estimated to be
about 60 , 000 dalton. The optimum temperature and pH for the
activity of the purified amylase were shown to be 70°C andd pH
5 - 9 respectively. The purified enzyme was les s s table at
higher temperature but 1 mM CaCl2 s tabilizes it significantly.
The purified enzyme has higher a f finity towards longer chain
dextrins and more complex substrates such as starch . Thin-layer
chromatography of enzymatic hydrolysis on various starches and
dextrins indicated that the purified amylase behaves similar to
that of a-amylase.
xii
Abstr ak tesis yang dikemukakan kepada Senat Uni vers iti Pertanian Mal ays ia sebagai memenuhi keperluan untuk Ijazah Master Sa ins .
Pengerusi
Fakulti
PENGHASILAN DAN PENCIRIAN AMILAS E TERMASTABIL DARI BACILI,US CIRCULANS YANG DIPENCILKAN
DAR! IOI,AM AIR PANAS
Oleh
SIIARIFAH SHAHRUL RABIAH BTE SYED ALWEE
SEPTEMBER 1992
Dr . Baharuddin Abdul Ghani
Sains Makanan dan Bioteknologi .
Dua s t rain bacteria penghasil ami lase telah dipenci lkan
dad kolam air panas dari Negeri Sembilan dan dikenalpast i
s ebagai Bacillus c i r c u l ans d a n s t r a i n-s t r a i n i n i t e 1 alt
dinamakan sebagai strain SB-l dan SB- 2 1 .
S u hu d a n p H o p t i m a u n t u k p e n g h a s i l a n a m i l a s e d a n
p e rtumbuhan m i k roorgani sma d i d a p a t i p a d a 5 5 ° C d a n p H 7 . 0 .
Enzim ini dihas i lkan dari pe rmulaan pertumbuhan dan mencapai
penghasilan yang maksimum pada 72 j am . Amil ase ini adalah enz im
yang separa aruhan dimana penghasi lan berlaku hanya apabila
t e rdap a t kanj i 1% . Kehadi ran m a l t o s a d a n m a l t o t r i o s a 20 mM
dapat meningka tkan lagi penghasilan enzim . Kehadi ran glukosa
didapati menindas penghas i lan enzim .
xii i
Enzim daripada Bacillus circulans strain 8B-l kemudianttya
ditulenkan melalui kromatografi turus pertukaran ion selepas
pemendakan ammonium sui fat 20-40\ dan penurasan ultra . Satu
puncak aktiviti dan 31 . 6\ hasil penulenan didapati dengan
faktor penulenan sebanyak 53 . 9 kali ganda . Dengan menggunakan
8DS-PAGE, enzim ini didapati homogen dan beberat molekul
dianggarkan 60,000 dalton . 8uhu dan pH optimum bagi enzim yang
ditulenkan ini adalah 70°C dan pH 5 - 9 . Walaubagaimanapun ,
enzim ini didapati kurang stabil pada suhu yang tinggi tetapi 1
mM CaClz dapat menstabilkannya . Amilase yang telah ditulinkan
ini mempunyai keafinan yang lebih tinggi terhadap dekstrin
berantai panjang dan subs trat kompleks seperti kanji.
Kromatografi lapisan nipis bagi hidrolisis enzim terhadap
berbagai jenis kanji menunjukkan bahawa amilase yang ditulenkan
ini bertindak seperti a-amilase.
xiv
CHAPTER I
INTRODUCTION
Amylases are classified as 'hydrolases' (BC 3. 2. l) and are
enzymes that catalyze the hydrolys is of O-glycosyl compounds
(Yamamoto, 1988) . They are divided into three main types namely
exo-acting, endo-acting or deb ranching enzymes according to
thei r mode of action. Exo-acting amylases degrade amylose and
amylopectin of sta rch by the success ive removal of low
molecular weight products f rom the non-reducing chain ends.
Rxo-acting amylase include �-amylase (Rxo-a-l, 4-g1ucan
maltohydro1ase, EC 3. 2. 1. 2) and glucoamy1ase (Exo-a-1, 4-g1ucan
glucanohydrolase, EC 3. 2. 1. 3) which produce maltose and glucose
respectively (Fogarty and Kelly, 1980) . Endo-acting amylase,
which is the mos t widel y occuring form of amylase, cons ists of
only a-amylase (Endo-a-I, 4-glucan glucanohydrolase, BC
3. 2. 1. 1 ) . It catalyzes the random hydrolysis of starch-type
polysaccharides, which results in a rapid reduction of iodine
blue value and viscosity of substrate depending on the
specific number of bonds broken. Compared with �-amylase or
glucoamylase, the reduction of molecular weight of subs trate is
much faster with a-amylase than ei ther �-amylase or
glucoamylase. Debranching enzymes which include pullulanase (EC
3. 2. 1. 41) and i soamyla se (EC 3. 2. 1.68) hydrolyse only a-l, 6-
glycosidic bonds.
1
a-Amylases are found in mammals, higher plants, fungi, and
bacteria . Bacteria are the most widely used and versatile
source of a-amylase . In addition, this source i s easily
amenable to genetic manipulation thus providing an opportunity
for the development of enzymes with more des i rable operational
parameters . To date most of the a-amylases used in industry are
produced by Bacillus sp . , especially Bacillus amyloli quefaciens
and Bacillus licheniformis. Bacillus sp . produce both
saccharifying and liquefying a-amylases which are
di s tinguishable by thei r mechani sms of s tarch degradation . For
example, Bacillus subtilis var . amylosacchariticus, Bacillus
subtilis Harburg and Bacillus natto all produce sacchari fying
a-amylase ( Hatsuzaki et al . , 1974) . Bacillus amyloliquefaciens,
on the other hand, p roduces large quantities of liquefying a
amylase (Welker and Campbell, 1967) .
Thermostable amylases have been isolated f rom various
strains of Bacillus spp . These include Bacillus
stearothermophilus ( Manning and Campbell, 1961) , Bacillus
licheniformis ( Horgan and Priest, 1981) , Bacillus coaqulans
( Hedda and Chandra, 1960) and Bacillus acidocaldarius
( Buonocore et al . , 1976) . Four interesting observations have
been made on the comparative biochemistry of thermos table
enzymes. First, the heat stability of these enzymes may be
associated with increased hydrophobic bonding at the core of
the molecule ( Doig, 1974 ) . Second, the thermostable a-amylase
was found to have an unfolded s tructure as opposed to a more
3
typical globular structure of the mesophilic enzyme ( Manning
and Campbell, 1961) . Third , an increase in hydrogen bonding may
be involved in heat stability. Finally , these enzymes appea r to
lack cysteine residues and as a consequence cannot contain
disulfide linkages (Amelunxen , 1967) .
Alpha-amylases have numerous biotechnological
applications , for example, in starch conversion proces ses a
amylase are used to produce syrups containing oligosaccharides ,
maltose and glucose , in the brewery , food and textile
industries . Bioconversion of starch into sugars and other
alternative food products makes up the major part of the
starch-processing industry. In the industrial production of
glucose syrup, gelatinized starch is subjected to hydrolysis by
a-amylase to produce dextrins. This process is called
'liquefaction'. The dextrins are then hydrolysed by
glucoamylase in the ' saccharification' process to produce
glucose (Swinkel , 1986) .
The enzymatic hydrolysis of starch is widely used because
it offers many advantages over the older technology of acid
conversion . Enzymes are more specific and efficient as
catalysts when compared to acid. According to Swinkel (1985) ,
the enzymatic process resulted in the following benefits : 1)
by-product formation is reduced more than IO-fold; 2) the
finished syrup only contain half the ash content; 3) less color
formation and easier refining; 4) Na+ and Cl- level is reduced
4
5-fold; 5) lower quality starch can be used: and 6) lower
energy costs.
Thi s project was done mainly for academic reasons, to
learn about local amylolytic thermophilic bacteria and the
enzymes produced by them. The knowledge gained in thi s study
would further contribute towards the utilization of local
substrates by biotechnology.
CHAPTER II
LITERATURE REVIEW
S tarch The Substrate
Starch occurs as the major reserve carbohydrate in all
higher plants in the form of water insoluble granules. It is
readily as similated in the human diet; in fact , a very high
p roportion of the world food intake is s tarch. Microscopic
examination reveals that starch is composed of tiny, white
granules, ranging from about 2 to 100 um in diameter. The size
and shape of the granules are peculiar to each variety of
starch (Table 1) . other than cellulose, starch is the next most
abundant compound synthesized by p lant cells. It is a
'renewable' substance; a new supply of starch is grown annually
(Swinkel, 1985) . 'The properties of starch vary with the plant
source f rom which it is derived.
Starch is a polymeric carbohydrate, composed of C , H and 0
atoms in the ratio 6:10:5. CCsHloOe)n. It is considered to be a
condensation of glucose polymers. The glucose units are present
as anhydroglucose units. If starch is treated with acids or
certain enzymes, it is broken down into its constituent glucose
molecules. The glucose units are linked to one another through
the Cl oxygen as glucoside bond. The glucoside bonds are stable
under alkaline conditions and hydrolyzable under acid
5
6
Table I
Properties of Starch Granule
Starch Type Size Range Shape ( Diameter)
( pm)
Corn Cereal 3 - 26 Round, Polygonal
Potato Tuber 5 - 100 Oval, Spherical
Wheat Cereal 2 - 35 Round, Renticular
Tapioca Root 4 - 35 Oval, Truncated
Rice Cereal 3 - 8 Polygonal, Angular
S ago Pith 5 - 65 Oval, Truncated
Source : Swinkels (1985)
conditions. The glucose unit at the end of the polymeri c chain
has a latent aldehyde group and is known as the reducing end
group .
Most starches are a mixture of two types of polymers : (1)
a linear cha in molecule termed amylose and (2) a branched
polymer of glucose termed amylopectin. Each of these polymers
have a wide range of molecular sizes. Starches of different
origin have different amylose and amylopectin ratios ( Table 2) .
Amylopectin made up about 75-85\ of starch.
Amylose, the linear polymer, contained up to 6000 glucose
units and is connected by a-I, 4-glucos idic linkages ( Figure 1 ) .
Enzyme studies also indicated a trace amount of branching in
Table 2
�ylose + A.ylopectin contents of Various Starches
S tarch
Corn
Potato
Wheat
Tapioca
Rice
Sago
Waxy ma ize
Amylose (!Its)
28
21
28
17
17
27
0
CHptt
OH
Amylopectin (!Its)
72
79
72
83
83
73
100
H OH
Ave . DP Amylose
800
3000
800
H OH
Rgure 1. Structure of Amylose Molecules
Ave. DP Amylopectin
2, 000 , 000
2, 000 , 000
2 , 000, 000
7
8
the amylose molecul e . The branched amylose may contain 3 to 20
chains, with an average chain length of about 500 glucose
units . The degree of polymerization (DP) of amylose covers a
wide range depending upon the source of the starch (Swinkel,
1985) . For example, the amylose molecules of potato and tapioca
starch have a substantially higher molecular weight than maize
and wheat starch amylose .
Amylose forms inclusion complexes with iodine and various
organic compounds such as butanol, fatty acids,
phenols and hydrocarbons. These complexes are
surfactants,
insoluble in
water. It is believed that amylose complexes by forming a helix
coil around the complexing agent (Swinkel, 1985). The complex
of amylose with iodine gave a characteristic blue colour,
which is used to establish the presence of amylose-containing
starch .
Amylopectin (Figure 2) is a highly branched structure
consisting of short linear chains with a DP ranging from 10-60
glucose units, with an average of about 22 . The glucose units
are connected to each other by a-l,4- and a-l,6-g1ucosidic
linkages . The glucose units with an a-l, 6-glucosidic linkages
are the branching points which cause the interlinkages of the
glucose residues that gave rise to a ramified or bushlike
structure of the amylopectin molecule. The branching points
make up about 5\ of the total glucose unit in amylopectin .
Amylopectin is one of the largest molecules in nature with an
average DP of about 2 mil lion . The molecular weight of
9
Figure 2. Structure of Amylopectin Molecules
amylopectin is about I 000 times as high as the molecular
weight of amylose (Swinkel, 1985) .
Starch granules are insoluble in water below 500e
(Fogarty , 1983) . When a suspension of starch in water is heated
beyond a cri ti cal temperature, the granule will swell many
times i ts original size. The critical temperature also known as
gelatinization temperature, va ries f rom 55°e to 800e depending
on the type of starch. When heating is continued , the swollen
starch granules will disintegrate into a viscous paste which is
the swollen starch aggregates. The process of transformation
f rom starch into the viscous starch paste is called
gelatinization. This hydrocolloidal property of starch makes i t
suitable for a great variety o f applications. starch and i ts
derivatives are widely used in the manufacture of foods,
paper, textiles, adhesive, pharmaceuticals and building
materials. Half of starch produced is used to produce syrups
and sugars .
