9

REVIEW OF LITERATURE
Starch degrading amylolytic enzymes is of great importance in biotechnological
application ranging from food, fermentation, and textile to paper industries etc. Alpha
amylase is a key enzyme in metabolism of spacious diversity of living organisms which
utilize starch as carbon and energy sources. It can hydrolyze starch, glycogen and
related polysaccharides by randomly cleaving internal -1,4-glucosidic linkages to
produce different sizes of oligosaccharides. Amylases are enzymes which hydrolyze the
starch molecules in to polymers consists of glucose units. Alpha amylase is ubiquitous
in distribution, with plants, bacteria and fungi being the major sources. Most of the
microbial alpha amylases belong to the family 13 glycosyl hydrolases, and they
contributed numerous common properties. But different reaction specificities have been
observed across the family members. Structurally alpha amylase possesses barrel
structures and is responsible for hydrolysis or formation of glycosidic bonds in the conformation. Stability of alpha amylase has extensively been studied; pH and
temperature have very vital roles to play.
Alpha amylase acts on starch and breaking them up into sugars (hence the term
saccharification). Starch is a carbohydrate source consisting of two molecules amylose
and amylopectine. Amylose is formed from chains of glucose linked 1,4 and
amylopectine is formed from 1,4 linked chains of glucose with 1,6 linked branch
points. The amylases are enzymes that work by hydrolyzing the straight chain bonds
between the individual glucose molecules that make up the starch chain. A single
straight chain starch is called an amylose. A branched starch chain (which can be

10
considered as being built from amylose chains) is called an amylopectin. These
starches are polar molecules and have different ends.

Alpha amylase can be derived from several sources such as plants, animals and
microbes. The microbial enzyme meets the industrial demands a large number of them
are available commercially and have almost replaced chemical hydrolysis of starch
processing industry (Pandey et al., 2000). The major advantage of using
microorganisms for the amylase production is economical bulk production capacity
and microbes are also easy to manipulate to obtain enzymes of desired characteristics
(Lonsane and Ramesh, 1990). Alpha amylase has been derived from several fungi,
yeasts, bacteria and actinomycetes, however, enzymes from fungal and bacterial
sources have dominated applications in industrial sectors. Fungal sources are mostly
terrestrial isolates such as Aspergillus species. Mode of action, properties and product
of hydrolysis differ, some what and depend on the source of enzyme. Two types of
enzymes have been recognized called as liquefying and saccharifying. The main
difference between them is that the saccharifying enzyme produces a higher yield of
reducing sugar than liquefying enzyme. Many scientists carried out extensive work on

11
alpha amylase production. The enzyme production is dependent on the type of strain,
composition of media and methods of cultivation. Generally fungi secrete alpha
amylase (dextrinizing enzymes) although a few fungi have been known to secreted
alpha amylase and beta amylase (saccharifying enzymes). A. oryzae EI 212 secrete
alpha and beta amylase or both depending upon the composition of media and
fermentation conditions. The nature and amount of extracellular amylase produced by
Aspergillus species determine the efficiency of conversion of starch to
oligosaccharides.
Tokhadze et al. (1975) isolated 86 strains of the Aspergillus producing
maximum acid stable alpha-amylase. Repeated cultivation of the selected strains in
the Minoda agar medium along with sodium nitrate during submerged cultivation
showed a 3-fold increase in the alpha amylase production. Yabuki et al. (1977) studied
rapid induction in the alpha amylase production by A. oryzae using inducer such as
maltose. The mycelia were taken from 20 h old cultures and cultivated on the medium
containing peptone and glycerol. Afterwards these cultures were starved for 5 h; in
this case maltose was added as inducer. During first hour of induction, both extra and
intracellular alpha amylases were produced with the same rate (70-80/g of cells/h).
After 1.5 h remarkable increase in alpha amylase production takes place and enzyme
production reached at optimum rate. No significant increase was occurred in the
weight of mycelia during 2 h of induction. When the purified samples of these intra
and extracellular enzymes were tested by using diethylaminoethylcellulose column
and techniques of gel filtration, both enzymes were showed similar properties in all
respects. Vallier et al. (1977) observed alpha amylase production after the lysis of

12
mycelia. For this purpose, mineral medium was used which consist of starch and
glucose. The lysis of mycelium seems to be due to the action of hydrolyzing enzyme
dextranase and levulanase on the cell wall. The pH of the media has great impact on
the lysis of cell wall and alpha amylase secretion. With increase in the pH of mineral
medium up to 8.8 the secretion of enzyme and lysis of mycelial wall were greatly
increased. This method makes it easy to get 3 times more enzyme production.
Sinha and Chakrabrty (1978) reported Aspergillus wentii hydrolysed the soluble
starch in to maltose. The optimum amylase production by using A. wentii was obtained
when fermentation medium consisted of Tryptophan as nitrogen source along with 1
% starch which was incubated for 72 h at 20C and pH of medium was adjusted at 6.
The enzyme activity was greatly inhibited with the addition of 1mM sodium
iodoacetate. However, enzyme production was increased 3.51 to 6 mg/ml with the
addition of 10 mM sodium citrate. Varnavskaia et al. (1978) studied the impact of pH
on the protein conformation and alpha amylase activity produced by using Aspergillus
terricola. Dispersion of optical rotation technique showed that macromolecule of
alpha amylase consists of alpha helix and beta structures. The change in the values of
pH resulted in two conformational forms. When decrease in pH occurred from 4-2
alpha helix structure uncoiled and degradation of beta forms occurred with the
increase in the pH from 8-12.
Mahmoud et al. (1978) reported the use of different agricultural by-products and
wastes such as wheat bran, rice bran, cane molasses, corn bran, glucose syrup, corn
starch as a substitute of original carbon source in the fermentation medium for the
synthesis of alpha amylase by Aspergillus niger NRRL-337. The medium containing

13
rice bran showed maximum alpha amylase activity. The nitrogen source also
substituted by such type of material that makes the medium economic such as corn
steep liquor, corn steep precipitate, dried yeast and gluten-30 and 50. Corn steep
precipitates give highest alpha amylase production compared to other nitrogen
sources. From these results, it was concluded the medium containing rice bran 7.2 %,
corn steep precipitate 2.5 %, magnesium sulfate 0.1 %, potassium di hydrogen
phosphate 0.1 % and calcium carbonate 0.1 % showed maximum activity. The fungal
amylase was isolated and purified from this medium. The purified enzyme showed
optimal activity at 40C and pH 4.3. Allen and Thoma (1978) studied alpha amylase
produced from A. oryzae acts on reducing ends, and maltotriose which was uniformly
labeled. The enzyme breaksdown the glycosidic bonds during enzyme substrate
formation.
Augustin et al. (1981) examined the activity and production of alpha amylase
and alpha glucosidase in the some members of ascomycetes, imperfect and mucoral
fungi. The factor of polysaccharide system which was responsible for the consumption
of alpha(1 to 4) glucans was described along with screening of the growth of
organism or fungi on soluble starch. Forty nine strains were tested for the production
of amylolytic activity and only twenty nine strains showed this activity. Kasim (1983)
investigated the biosynthesis of alpha-amylase and amyloglucosidase (EC.3.2.1.3) by
A. oryzae in submerged fermentation. For this purpose different sources of carbon and
nitrogen were tested. The medium which shows maximum production of alpha
amylase and glucoamylase was not very costly and consists of following components
in (%) corn steep liquor 3, magnesium sulfate 0.1, potassium dihydrogen phosphate

14
0.1, defatted rice bran 8 and calcium chloride 0.1. The pH of medium was adjusted at
5. The optimum conditions for enzymes production were incubation at 28C for 96 h
and the inoculum consists of 0.5 % mycelial suspension. Erratt et al. (1984) reported
that starch was used as inducer for alpha amylase production from the A. oryzae.
When glucose was used as carbon source the production of both intra and extracellular
amylase was very low. While starch was used as carbon source increase in the activity
of alpha amylase was noticed. In glucose grown cultures intracellular activity of alpha
amylase increased 6.5 fold; however, 20 fold increase was observed in extracellular
activity. Regardless of type of carbon source used, the active protein react only those
antibodies which showed specificity only for alpha amylase and active protein have
molecular weight 52 500 +/- 1800.
Ustiuzhanina et al. (1985) studied the regularities in the biosynthesis of protease
and alpha amylase by using washed cells of selected strain of A. oryzae. The results
enabled us to compare the constitutive characters of protease and alpha amylase by
selected strain of A. oryzae. Carbon, nitrogen and sulfur play very important role in the
regulation of protease synthesis. However, in alpha amylase synthesis, merely carbon
source played an important role. Phosphorous was vital for the synthesis of both alpha
amylase and protease. Removal of phosphorous from the medium adversely affects the
production of both enzymes. The alpha amylase and protease production was
stimulated by the addition of celatin.
Hayashida and Teramoto (1986) reported that a protease negative mutant M33 of A.
ficum was obtained by treating A. ficum with MNNG. This strain showed highest alpha
amylase activity compared to parent strain in submerged fermentation at optimal

15
condition i.e. 30C for 24 h. The molecular weight of purified enzyme was 54, 0000.
MacGregor (1988) studied two computerized methods which explain the sequence of
amino acid in the secondary structure of protein in alpha amylase which was produced
by A. oryzae. Alpha-amylase produced by A. oryzae, showed three dimensional
structures. The computerized methodology explained the position of amino acid and
gave the predictions about the structure of alpha amylase from different sources. It
was noticed all alpha amylase having known amino acid sequence possess same basic
structure, these alpha amylase possess barrel shape structure which was surrounded by
eight helices. The strong resemblance were found in those part of protein which take
part in binding the Ca+2 ions and active site of enzyme which play important role in
catalyzing the substrates hydrolysis. The active site was composed of amino acids
which were specifically found in the loop joining the adjacent helix. The changes in
the length and sequence of amino acid created the differences in binding the substrate
and produced modifications in the action pattern of alpha amylase from different
origins.
Ali and Abdel-Moneim (1989) reported that the best temperature for the
preservation of A. flavus var. columnaris alpha-amylase was -5C followed by 5C.
CaCl2 at 0.005 M had no effect on the activity in both temperatures. Repeated freezing
(-5C) and thawing followed by freezing (-5 C) had no effect on stability of alphaamylase. On the other hand, 25C was the lowest preservation temperature without
any effect on the stability of alpha-amylase. 0.005 M CaCl2 decreased the activity of
alpha amylase and reached a 100 % inhibition at 35th day. The fungal alpha amylase
had an optimum temperature of 55C at pH 4.6, but had 60C in buffer containing

16
0.005 M CaCl2 and 50C in buffer containing 0.005 M Na2-EDTA. The addition of
0.01 M CaCl2 greatly increased the thermostability of alpha amylase at 40, 45, 50, 55
and 60C for 30 min. Optimum pH for alpha amylase was 5, but in the presence of
0.01 M CaCl2 or Na2-EDTA 5.6. The enzyme was only stable for 4 h at 25C.
Whereas, addition of 0.01 M CaCl2 showed a loss of 4 % compared to a 22 % loss in
the presence of 0.01 M Na2-EDTA after 4 h at 25C and 65 % loss in the presence of
0.01 M CaCl2 together with 0.01 M Na2-EDTA in the beginning and a 100 % loss
after 4 h at 25C. The optimum temperature for the activity of alpha-amylase at pH 5
was 50C for the enzyme only but 55C in the presence of 0.01 M CaCl2. However, at
pH 6 and 7 optimum temperature was 55C for the activity of the enzyme only or with
0.01 M CaCl2. The presence of 0.01 M CaCl2 at pH 5, 6 and 7 resulted in increase of
enzyme activity at the temperatures above 50, 40 and 25C, respectively. However,
0.01 M CaCl2 at pH 5 and 6 resulted in decreasing enzyme activity at temperatures
below 55 and 45C, respectively.
Rousset and Schlich (1989) screened different species of A. niger for the
synthesis of amylolytic enzymes i.e., alpha amylase and glucoamylase by using the
submerged fermentation. Statistical analysis was used to explain the behaviour of
culture instead of explaining optimization of fermentation conditions. Principal
component analysis (PCA) was used to explain the affect of three agitation rates on
amylase production and the formation of many other factors which affect the growth
in indirect way. The result of Principal component analysis (PCA) describes the
transfer of oxygen at different agitation rate influences enzyme production and carbon
dioxide. The production of carbon dioxide was indirect growth measurement.

17
Maximum alpha amylase production was obtained at lower agitation speed while in
case of glucoamylase intermediate agitation speed gave maximum alpha amylase
production. Shah et al. (1991) optimized the conditions for the synthesis and recovery
of alpha amylase from A. oryzae. A. oryzae alpha amylase retained 100 %, 61 % and
58 % respectively when preserved for 12 months at 4C, ambient temperature and
37C. Harway (1991) has isolated thermophilic bacteria from the soil which was
preliminary enriched with 0.6 % starch broth at 55C. These bacteria had ability to
hydrolyze the starch. Of the entire isolated cultures one was Bacillus coagulans, which
was best producer of alpha amylase. The maximum production was obtained in
optimal condition which consists of incubation temperature 55C, 200 rpm agitation
speed, 48 h incubation period and broth extract starch agar medium.
Tsekova et al. (1993) studied the ability of Aspergillus genus for alpha amylase
production. When 3 % soluble starch was used in Czapek-Dox agar and in liquid
Czapek-Dox media maximum alpha amylase production was obtained. Sudo et al.
(1993) studied the fermentation medium containing all the components which were
necessary for the production of acid stable alpha amylase (asAA) by A. kawachii using
submerged fermentation. One hundred and thirty milligram of acid stable alpha
amylase per liter of medium was produced after 5 days of inoculation at 30C in
submerged fermentation. Glycogen was present as stored polysaccharide. When the
amount of stored glycogen (CSG) decreased and inducer such as dextrin was present
synthesis of acid stable alpha amylase started. Maximum production of as AA was
obtained when amount of CGS reaches at zero. When the amount of CGS increased
production of acid stable alpha amylase tend to be decreased. The amount of glucose

18
in the medium and growth of mycelia was strongly influence by the concentration of
CGS. The quantity of acid stable alpha amylase was directly proportional to the
production of mycelia. Soccol et al. (1994) tested two species of Rhizopus for protein
enrichment of both cooked and raw cassava and also for the synthesis of
amyloglucosidase and alpha amylase in solid and submerged fermentation. The
protein enrichment and maximum enzyme synthesis were obtained in solid state
fermentation. Cooked cassava showed optimum production in solid state fermentation;
however, maximum synthesis of amyloglucosidase by R. oryzae was obtained when
raw cassava was used. Khoo et al. (1994) achieved fifty units per milliliter amylolytic
activity by using A. flavus in liquid medium containing topica starch. The culture
filtrate was subjected to electrophoretic analysis. This analysis showed filtrate contains
only one type of amylolytic enzyme named alpha amylase. The following factor
support the identification of alpha amylase (i) iodine stained starch quickly become
colourless (ii) starch digestion resulted in the formation of a mixture of glucose,
maltose, maltotriose and maltotetrose. Purification of enzyme was involve the use of
ammonium

sulfate

precipitation,

ion

exchange

chromatography

and

gel

electrophoresis. The purified enzyme showed 52.5 2.5 kDa molar mass with an
isoelectric point at pH 3.5. Characterization of enzyme showed the maximum activity
of purified enzyme was noticed at pH 6 and 55C.
Omori et al. (1994) isolated acid labile alpha amylase (A-3) from A. kawachii in
barley koji. The enzyme was purified by using the different techniques such as ion
exchange chromatography and gel filtration. The changes in new alpha amylase
production was compared with two known alpha amylase represented as A-1 and A-2.

19
Sodium dodecyl sulfate poly acrylamide gel eletrophoresis showed A-3 has molecular
weight 56,000. The enzyme showed constant production at 40C approximately for 54
h. However, in traditional method formation of A-3 was not detected after 36 h. In the
presence of 2 % citric acid in barley A3 was formed upto 36 h. The results indicated
production of A3 was influenced both by temperature and initial concentration of
citric acid. Chang et al. (1995) reported that alpha amylase produced by A. oryzae
was purified by passing through the different steps in a specific sequence such as
amylopectin affinity adsorption, DEAE-Sepharose ion exchange chromatography and
sephacryl S-200 HR gel filtration. After passing through these steps the enzyme
showed 16 fold increase in the purity and 45 % of enzyme was recovered. The
optimum conditions for purified enzyme was pH range 4-5, temperature 50C and km
value for starch hydrolysis was 0.22 %. Incubation for 30 min at 50C result 80 % lose
in enzyme activity. The heat denaturation constant and molecular weight by gel
filtration was 0.024/m and 52 kDa, respectively. The enzyme activity was inhibited by
using Mercuric ion (0.3m M), DNFB# (6mM), NBSI (6mM) and NAI (6mM). The
hydrolysis of maltoheptaose by the enzyme resulted in the formation of maltotriose
and maltotetraose.
Donmez and Melike (1996) isolated bacteria showing amylolytic activity from
different samples and grouped them on the basis of showing amylolytic activity in the
solid and liquid fermentation media. Of all the isolated strains Bacillus subtilis
produce 24 U/ml alpha amylase. Different carbon sources were added to the
fermentation media to check effect of these carbon sources on alpha amylase
production. The maximum activity of alpha amylase 360 U/ml was obtained in the

20
presence of dextrin and optimum temperature was 50C for enzyme production.
However, if enzyme was incubated for 2 h at 100C 23 % of this activity was lost.
Carlsen et al. (1996) tested the stability of alpha amylase produced by A. oryzae at
different pH. The enzyme showed highest stability at neutral pH (5-8); however,
beyond this pH range a great loss in the activity of enzyme was noticed. On line FIA
system was used and a rate constant was obtained by the empirical expression k = 1.19
107 [H+]1.99 (h1) explained the inactivation of enzyme was greatly influenced by pH
values. The inactivated enzyme again obtained some of its activity at pH 6 and this
reactivation steps also obey the first order kinetics rules. The contamination of
protease in the protein sample was not result to the irreversible loss of activity.
Abou Zeid (1997) isolated filamentous fungi from cereals and screened to test
the alpha amylase producing potential. The strain which showed highest ability for
alpha amylase production was identified as A. flavus. The enzyme was purified by
using starch adsorption methodology. The polyacrylamide gel electrophoresis (PAGE)
indicated the molecular weight of A. flavus was 75, 000 3,000. The optimum
temperature for purified enzyme was 7 and 30C, respectively. The use of potassium
ions increased the activity of alpha amylase. However, magnesium ions did not
extremely influence the enzyme activity. The activity of alpha amylase was greatly
inhibited in the presence of manganese, zinc, copper and ferric ions. The hydrolysis of
native starch by A. flavus resulted in the formation of glucose and some other
oligosaccharides
Kajiwara et al. (1997) studied the production of acid stable alpha amylase from
A. kawachii during production of shochu-koji. From barley shochu-koji two types of

21
the acid stable alpha amylase (as AA) represented as as A-1 and as A-2 were puified.
The asA-1 and asA-2 showed different adsorption characteristics on raw starch. The
activity of as A-1 slowly increased during the process of shochu-koji production but
dropped after incubation of 24 h. Contrary to as A -1 the activity of as A-2 was
increased with increase in the incubation time. Temperature affect ratio of as A1 to
total as AA activity. When acid protease and as A-1 were incubated along with each
other and this sample was analyzed by SDS-PAGE. A known band was appeared in
the place of as A-1 band after 12 h of incubation. The unknown protein showed all the
characteristics which were present in as A-2. The result showed acid stable alpha
amylase was found in different form just like the glucoamylase produced by A.
awamori during the production of shochu-koji. Spohr et al. (1997) examined alpha
amylase producer strain of A. oryzae for the production of recombinant protein and
affect of growth on the production of protein. The comparison of these strains for
morphology and impact of morphology on the protein indicated the mutant strain
having denser mycelium, produce more alpha amylase compared to other strains.
Arnesen et al. (1998) cultivated thermophilic fungus in the presence of dextran
(having low molecular weight) along with Tween 80 or Triton X-100. The
fermentation was carried out in shake flasks for more than 120 h. The 2.7 fold increase
in the activity of alpha amylase was observed in medium containing Tween 80
compared to the medium with out Tween 80. The medium containing Tween 80
showed increase in the alpha amylase production after 48 h; while general protein
secretion was stimulated after 24 h of inoculation. The Tweeen 80 also influences the
production of biomass. The production of biomass increased gradually with the

22
increase in the concentration of Tween 80. Contrary to this Triton X-100 produce
reverse effect. It was noticed increase in the amount of Tween 80 resulted greater than
3 fold increase in the total extracelluar protein. The Tween 80 had no effect both on
the hyphal length and diameter. Glycosylation degree was also not effected by the
Tween 80. Anidyawati et al. (1998) purified three forms of alpha amylase to
homogeneous state by using the methodology of column chromatography. These
forms of alpha amylase were produced from A. awamori. These forms were
designated as Amyl1, Amyl 11, and Amyl 111. The SDS PAGE indicated these three
forms possess 49,000, 63,000 and 97,000 molecular weight, respectively. The
optimum pH for Amyl 11 and Amyl 111 was 5.5 while in the case of Amyl 1 the pH
was 4. Maltose and maltosetriose were formed by the hydrolyzing action of Amyl 1
on malto-tetraose-pentose,-hexaose,-heptose and and -cyclodextrin. However Amyl
1 produces no hydrolyzing effects on raw corn starch, maltose, maltotriose,
isomaltotriose, isomaltosse, and -cyclodextrin. Unlike Amyl 1 both Amyl 11, and
Amyl 111 have ability to hydrolyze maltotriose, raw corn starch and alpha, beta,
gamma cyclodextrin resulting in the formation of maltose along with minor products
of glucose and maltose. The range of soluble starch hydrolysis through Amyl 1, Amyl
II and Amyl III was 33, 35 and 38 %, respectively.
Jin et al. (1998) used A. oryzae for alpha amylase production and microbial
biomass protein (MBP) from starch processing waste water (SPW) in air lift
bioreactors. The production of MBP and fungal alpha amylase was carried out under
the optimized conditions i.e., pH 5 and 35C. Bioproduct yield obtained from 12h
batch culture was 6.1 g/l. This yield consists of 55 EU/ml of alpha amylase and 38 %

23
protein. The enzyme showed stability at pH 5-9 and 25-35C. Spohr et al. (1998)
tested three different strain of A. oryzae having the ability to form recombinant protein
with respect to growth and alpha amylase production. One was wild strain and the
second strain was a transfomant strain which consists of additional copies of alpha
amylase gene while third strain was morphological mutant. It was observed the
production and growth of organism were correlated. Comparison of production and
morphology of these strains indicated the variations in the morphology had direct
impact on enzyme production in submerged fermentation.
Moreira et al. (1999) isolated a fungal strain from the soil having the ability to
produce amylolytic enzymes. This strain was identified as A. tamari. A. tamari formed
both alpha amylase and glucoamylase in the mineral medium concomitant with carbon
source i.e., 1 % starch or maltose. The formation of alpha amylase and glucoamylase
indicated tolerance to wide range of initial fermentation medium pH (4-10) and
temperature (25 - 42C). Ion exchange chromatography was used for the separation of
alpha amylase and glucoamylase. Partially purified alpha amylase and glucoamylase
showed maximum activities at pH 4.5 and 6 and stability at pH 4-7. The temperatures
at which enzymes showed highest activities was between 50 - 60C.
Pedersen and Nielsen (2000) reported the effect of organic and inorganic
nitrogen sources on alpha amylase production by A. oryzae in continuous cultivations.
Both nitrogen sources were tested along with glucose. In case of inorganic nitrogen
source ammonia was better than nitrate. The comparison between organic and
inorganic nitrogen sources indicated organic nitrogen for example yeast extract or
casine hydrolysate was superior to ammonia. In the presence of 0.05 g/l casine

24
hydrolysate 35 % increase in alpha amylase production was found. The transcription
of the alpha amylase genes were not involved in the increase production of alpha
amylase the basic reason was the grater secretion of alpha amylase from the biomass.
Nguyen et al. (2000) optimized the composition of fermentation media for increasing
amylases production through Thermomyces lanuginosus by using the different ways.
The influence of different carbon and nitrogen sources was tested. The carbon and
nitrogen sources, which proved to be good substrates for the growth of T. lanuginosus
and exbhited maximal alpha amylase (92-125 U/ml) and glucoamylase (6-13 U/ml)
activites included starch, maltodextrin, dextrin, maltose, amylopectin, glucose dextran
and L-asparagine. L-asparagine at the level of 6.5 % was good for alpha amylase
production and 2 % L-asparagine was optimum for glucoamylase production. The pH
of medium was adjusted by using hundred millimolar citrate buffer for amylases
production. Response surface method (RSM) was used to find out the suitable
concentration of medium component for the synthesis of amylolytic enzymes. A
second order polynomial model was used at significance level 95 % (p<0.05) for alpha
amylase and glucoamylase. The selected composition of media was tested with respect
to synthesis of amylolytic enzymes.
Mariani et al. (2000) studied impact of Amaranth seed meal and the aeration on
the productiviy of alpha amylase by A. niger NRRL 3112. The assays for the selection
of fermentation media was carried out by using the rotary shaker at 250 rpm and 2.5
cm stroke. The selection of aeration conditions were carried out in New Brunswick
mechanically stirrer fermenter A fermentation medium containing 5.0g/l Amaranthus
cruentus seed meal produce 2750 U.Dun/ml alpha amylase with dry weight of 8.0 g/l

25
after 120 h, of inoculation. The optimum condition for alpha amylase production in
fermenter were fermentation period of 120 h, agitation rate 300 rpm and an air flow of
11/l/ min in limited concentration of dissolved oxygen. Although increase in agitation
speed increases the dissolve oxygen but it was not suitable for the formation of alpha
amylase. Morphology of A. niger such as long and branched hyphae was very
important to obtain the maximum alpha amylase production. Petrova et al. (2000)
reported the purification of wild and mutant strains of Thermomyces lanuginosus
ATCC 34626 a thermophilic fungus. The purification was carried out to homogeneity
by using the different techniques in a sequence such as precipitation with ice cold
propanol, anion exchange and molecular sieve chromatographic methods. The SDSPAGE results indicated purified alpha amylases (both with PI values of 3.0) have
molecular mass 58 kDa. The optimum pH for the activity of wild and mutant strains
was 5 and 4.5, respectively. 1 Cyclohexyl - 3 - (2-morpholinyl 4 - ethyl) carbodiimide (40 100 mM) and N- bromo succinimide (0.1 1mM) produce
inhibitory effect on the enzymes activity due to the presence of carboxylic groups and
tryptophan residues in the catalytic process.
Madihah et al. (2000) isolated and partially purified alpha amylase from the
fermentation of sago starch to solvent by C. acetobuylicum P262. The characterization
of partially purified enzyme showed the following optimal conditions. The highest
activity of alpha amylase was observed at pH 5.3 while enzyme showed stability from
pH 3-9. The highest activity of alpha amylase was found at 40C; however, if alpha
amylase was placed at 60C for 60 min merely 50 % of its original activity was
retained. The Km and Vmax values of alpha amylase for soluble starch were 0.31 g/l and

26
10.03 U/ml, respectively. Viswanathan and Surlikar (2001) designed the medium by
the use of fractional factorial method and Plackett-Burman design to study the
influence of component of Amaranthus paniculatas (Rajgeera) medium on alpha
amylase production by A. flavus. Fifteen components were used in developing the
medium. Out of these components only four i.e., CSL, NaCl, CaCl2 and NH4HPO4
were choosed on the basis of contrast coefficient values and selected as independent
variables for the Box-Behnken design. By using SPSS/PC +(version 7.5) statistical
analysis a polynomial multiple regression model was prepared. CSL, NaCl, CaCl2 and
NH4HPO4 increased the yield up to 81.3 % however, NaCl, CaCl2 influence the
product to the tune of 68.3 %. The comparison of control and optimized medium
exhibited 8 fold increase in production of enzyme in the optimized medium.
Carlsen and Nielsen (2001) tested the effect of different carbon sources such as
fructose, galactose, mannitol, glucose, glycerol, sucrose, and acetate on alpha amylase
production by A. oryzae in carbon limited chemostat cultures. A. oryzae was not able
to grow on such a medium which contain galactose as only carbon sources; however, a
combination of glucose and galactose allow the fungal strain to grow and produce
alpha amylase. Medium containing maltose and maltodextin indicated more alpha
amylase production during growth of A. oryzae compared to medium containing
glucose concentration less than10 mg/l. Sucrose, glycerol and mannitol showed low
alpha amylase production. Acetate alone did not show any production of enzyme but
acetate along with little quantity of glucose exhibited alpha amylase production. It was
observed alpha methyl-D-glucoside was acted as an inducer for alpha amylase
production but it was not as good as glucose.

27
Agger et al. (2001) evaluated the influential impact of formation of biomass on
the synthesis of alpha amylase by using the wild strain of A. oryzae and recombinant
strain of A. nidulans in submerged fermentation. It was noticed specific rate of alpha
amylase production was inversely proportional to the concentration of biomass
formation. When the concentration of biomass was increases 2-12 g dry weight/kg the
specific rate of enzyme production was decreased. However in case of recombinant
strain of A. nidulans in which gene creA was removed (which cause carbon catabolite
repression) no marked decrease in the specific rate of enzyme formation was observed.
The results indicated less alpha amylase production at high biomass formation was
due to slow mixing rate of vital components in viscous culture medium.
Ray (2001) isolated Penicillium sp possessing the ability to form alpha amylase
and xylanase in the presence of starch and xylan respectively, in fermentation. It was
noticed the optimum amylolytic activity and xylanolytic activity was obtained on 4th
and 6th day of fermentation respectively. The quality of alkalophilic strain of
Penicillium sp to hydrolyze starchy and hemicellulosic wastes made them a potent
strain for the large scale economic production of both enzymes using the cheap
substrates. Bogar et al. (2002) tested different strains of A. oryzae on spent brewing
grain (SBG) and corn fiber for alpha amylase production. A Plackett-Burman
experimental design was practiced to develop optimized media for alpha amylase
production using best producer strain. A. oryzae NRRL 1808 strain produced 4519 U
of alpha amylase/g of dry matter substrate in stationary 500 ml Erlenmeyer flask
culture after 72 h. The crude enzyme, in situ enzyme produced in solid substrate
fermentation material was economic biocatalytic product for animal feed and for the

28
production of bio alcohol from starchy substrate. Francis et al. (2002) investigated the
effect of spent brewing grains on alpha amylase production by A. oryzae NRRL 6270
when spent brewing grains utilized as sole carbon source. Maximum alpha amylase
production was obtained at 25C. At 30C almost similar results were obtained.
Optimum alpha amylase production [6870U/g dry substrate (gds)] was obtained in
solid state fermentation at 30C after 96 h by the use of suspension containing 1107
spores/ ml. Addition of any external carbon source in the spent brewing grain resulted
decreased in alpha amylase production.
Arnesen et al. (2002) used thermophilic fungus T. lanuginosus for alpha
amylase production in shake flasks. The fermentation medium contained carbon
source in the form of low molecular dextran. The fermentation was carried out up to
120 h. The results showed maximum alpha amylase activity after 96 h of inoculation
during stationary phase while the production of maximal biomass takes place after 48
h of fermentation. A same pattern was observed in the case of total extra cellular
protein. It was found many unidentified proteins and alpha amylase were de novo
synthesis by using pluse labeling techniques of proteins. The sequencing of alpha
amylase from T. lanuginosus using specific primmer and RT-PCR technique indicated
that transcription of alpha amylase was not start before the late growth phase and
reached at its highest value more than 24 h after maximum biomass was produced.
Gigras et al. (2002) used the central composite design along with 3 variables
i.e., starch, yeast extract, and di potassium hydrogen phosphate for alpha amylase
production by A. oryzae in shake flasks and bioreactor. The alpha amylase production
was 133U/ml in shake flasks while in case of bioreactor production was 161 U/ml.

29
However, there was great difference in the fermentation period of shake flasks and
bioreactor. The fermentation period for the maximum alpha amylase production by A.
oryzae in shake flasks was 120 h but in case of bioreactor this time period was reduced
to only 48 h. A high concentration of phosphate in the fermentation medium and use
of low inoculum size was essential to prevent the unnecessary foaming in bioreactor;
but managing the pO2 level and agitation rate was not compulsory for alpha amylase
production. The enzyme production increases with the increase in the pH of medium
and reached at its peak at pH above than 7.5. Thus in present study pH act as sign of
commencement or ending of the enzyme production.
Huang et al. (2003) developed a segregated model to explore the intrinsic
associations between growth, substrate consumption, cell differentiation and enzyme
formation by Bacillus subtilis in bioreactor. The segregated model represented three
different states of cell and the change from vegetative stage to sporangium and lastly
to mature spore. An age-based population balance model was used to explain the
maturity of sporangium in the direction of the formation of spores. Parameters in the
model were found out by placing the experimental data in the model. The model has
ability to describe the temporary behavior of B. subtilis in both batch and fed-batch
cultures.
Francis et al. (2003) optimized incubation temperature, initial moisture contents
and inoculum size by application of BoxBehnken design under the response surface
methodology for the highest production of alpha amylase by A. oryzae NRRL 6270.
The experimental data was added into a polynomial model to find out alpha amylase
production. A PlackettBurman design was used to test the influence of nineteen

30
nutrient components on alpha amylase production by the A. oryzae. Soybean meal,
CaCl2 and Mg SO4 were chosen on the basis of their positive affect on alpha amylase
production. A BoxBehnken design was used to select the best condition for alpha
amylase production. Incubation temperature 30C, initial moisture contents 70 % and
an inoculum size of 1107 spores/g dry substrate were the optimum conditions for the
alpha amylase formation by A. oryzae NRRL 6270 on SBG. Under selected conditions
of solid state fermentation SSF, about 20 % enhancement in enzyme production was
found. Kariya et al. (2003) purified alpha amylase from culture broth of A. oryzae
MIB 316. The enzyme had ability to efficiently hydrolyzed amylopectin, amylose and
starch and break down maltopentose to produce a maltotriose and maltose. However,
maltose did not produce glucose. The N-terminal sequence of first 10 residues and
many other molecular characteristics were similar to Taka-amylase.
Kusuda et al., (2003) isolated alpha amylase from an immobile culture filtrate of
Tricholoma matsutak. The enzyme was purified to homogeneity by sequential steps of
Toyopearl-DEAE, gel filtration, and Mono Q column chromatography. The alpha
amylase showed 3580 fold purity and 10.5 % recovery. SDS-PAGE analysis resulted
in a single protein band. The characterization of purified alpha amylase showed that it
was most active at pH 56 and having stability between wide range pH i.e., 410. The
experimental results also indicated the alpha amylase was somewhat thermostable and
showed thermostability at 50C while the optimal temperature was 60C. The sizeexclusion chromatography and SDS-PAGE showed that purified alpha amylase had
molecular mass 34 kDa and 46 kDa, respectively. The mercuric ion did not inhibit the
activity of enzyme. Measurement of viscosity, TLC and HPLC analysis indicated

31
amylases from T. matsutake was of endo type. The specificity of alpha amylase was
tested by using amylose along with various polysaccharides. This alpha amylase
rapidly hydrolyzed the -1,4 glucoside linkage in soluble starch and amylose A
(MW,2900), but was not able to hydrolyze the -1,6 linkage and cyclic
polysaccharides e.g - and -cyclodextrin.
Kanwal et al. (2004) extracted alpha amylase from Malus pumila (apple) by
homogenizing the apple in buffer for alpha amylase. After extraction, the enzyme was
purified by passing sequential steps of purification. The crude extract exhibited 3.09
U/ml alpha amylase activity was subjected to ammonium sulfate precipitation. This
partially purified enzyme produces 4.76 U/ml and showed 5.01 U/mg specific activity.
The enzyme was further purified by gel filtration chromatography (Sephadex G-150).
After gel filtration chromatograph it produces of 5.025 U/ml and specific activity
38.95 U/ml along with 20-fold purification. SDS-PAGE of enzyme removed the
undesirable proteins and single band of enzyme was appeared. Molecular weight of
alpha amylase was 51,180 D which was finding out by Sephadex G-150 column.
Amylase exhibited optimal pH 6.8, incubation temperature 37C, Km value 2.0x10-3
g/ml, max 540nm and incubation time for enzyme assay was ten min.
Apar and Ozbek (2004) studied the effects of temperature on the enzymatic
hydrolysis of starch from different sources such as corn, rice and wheat. Three
commercial alpha amylases produced from Bacillus sp. A oryzae and B. licheniformis
were employed for hydrolysis of starch. In every starch hydrolysis process, the
concentration of residual starch and the residual activity of alpha amylase in
percentage were determined at 50 and 60C temperature based upon the processing

32
time in a stirred batch reactor. Mathematical models were developed for using
experimental data of residual starch concentration from each source. Some
inactivation models were used to understand the relation between temperature and
stability of enzyme during hydrolysis of starch from enzymes having different origins.
El-Safey and Ammar (2004) reported that the amylolytic family has great
importance due to its wide spectrum of application. Alpha amylase produced from
Aspegillus flavus var.columinaris was isolated and characterized. The enzyme was
purified by using ammonium sulfate precipitation and Sephadex G200 filtration
method. The purified enzyme showed 9.97 fold purification and 6471.6 (units/mg
port/ml) specific activity. The alpha amylase activity amplified with the enhancement
of enzyme concentration. The optimum condition for the production of alpha amylase
was 0.2% (w/v) starch, while the optimal temperature was 35C. The purified alpha
amylase showed maximum activity at pH 6.2 after 30 h of incubation. Pimpa (2004)
reported that the highest alpha amylase production by Aspergillus sp. was obtained
after 24 h. Addition of suitable nitrogen sources and inorganic salts to the medium
appreciably increased the enzyme production. The maximum enzyme yield 36.5 U/ml
was obtained in the media containing wastewater, defatted soyabean 10 g/l, potassium
di hydrogen phosphate 10g/l, magnesium sulfate 5 g/l, zinc chloride 0.1 g/l. The alpha
amylase produced by Aspergillus sp. showed catabolic repression. The enzyme was
partially purified by subjecting into 60 % ammonium sulfate. The optimal pH and
temperature of partially purified enzyme was 5 and 50C, respectively.
Chavez et al. (2004) screened different carbon sources namely sorghum, soluble
potato, corn and cassava starches as well as maltose for the concurrent cultivation and

33
production of both alpha amylase and glucoamylase by a novel Trichoderma sp. even
though maltose gave better results compared to other carbon sources with respect to
activity of alpha amylase (about 28,000 U/l) and alpha amylase production (about 390
U/l/h), cassava and corn starches showed maximum glucoamylase activities (17,00018,000 U/l) and production of enzyme was almost similar to those obtained with
maltose (about 100 U/l/h). Because of its capability to produce both alpha amylase and
glucoamylase, the Trichoderma sp used in this study proved to be beneficial in a direct
process of raw starch saccharification with no preliminary gelatinization.
Konsula and Kyriakides (2004) isolated a somewhat thermophilic Bacillus
subtilis strain, from fresh milk of sheep possess the ability to produce extracellular
thermostable alpha amylase. The medium containing low starch concentration showed
maximum alpha amylase production at 40C. The enzyme exhibited highest activity at
135C and pH 6.5. The thermostability of alpha amylase increased in the presence of
calcium or starch. This thermostable alpha amylase was employed for the hydrolysis
of different starches. Ammonium sulfate crude enzyme preparations as well as the
cell-free supernatant actively break down the starches. The use of the clear supernatant
as enzyme source was highly advantageous mainly because it decreases the cost of the
hydrolysis. When the reaction temperature increased up to 70C, all of the substrate
showed higher rates of hydrolysis. Potato starch upon hydrolysis produced higher
concentration of reducing sugars compared to other starches at all tested temperatures.
Soluble and rice starch came at second and third position respectively, with respect to
reducing sugar liberating ability. However, in case of corn and oat starch alpha
amylase showed somewhat less affinity.

34
Ramachandran et al. (2004) investigated alpha amylase production by A. oryzae
in solid state fermentation (SSF).The substrate used was coconut oil cake (COC). Raw
COC was a good substrate and 1372 U/gds alpha amylase was produced in 24 h. As a
result of optimization of media component alpha amylase production was increased
(1827 U/gds) when solid state fermentation was carried out at 30C for 72 h along
with media contained 68 % moisture contents. Addition of glucose and 0.5 % starch
further increased the alpha amylase production (1911 U/gds). However, maltose
repressed the alpha amylase production. Alpha amylase production increased upon
adding the organic and inorganic nitrogen sources. When peptone at the level of 1 %
was added in the fermentation media 1.7-fold increase in enzyme activity (3388
U/gds) was observed.

The enzyme production and growth were correlated. The

activity became maximal when the fungal biomass was at its peak at 72 h.
Kunamneni et al. (2005) employed the response surface methodology to study
the collective impact of the nutritional parameters and to increase extracellular alphaamylase production in solid-state fermentation by T. lanuginosus. These nutritional
parameters consist of carbon source (soluble starch), nitrogen source (peptone) and a
concentrated mineral medium. A twenty three factorial central composite design using
response surface methodology was used to optimize the above three variables. The
best calculated values of these variables for optimal amylase production were soluble
starch 71.10 g/Kg, peptone 91.97 g/Kg and mineral salts solution 175.05 ml/Kg

with

an estimated alpha amylase activity of 5.085 105 U/Kg of wheat bran. These
parameters were checked in the laboratory and ultimate alpha amylase activity
obtained, 4.946 105 U/Kg of wheat bran, was very near to the calculated value.

35
Kiran et al. (2005) isolated the thermophilic Bacillus sp. K-12 from soil samples
having the ability to produced amylolytic enzyme. Effects of different carbon sources
and chemicals on production of alpha amylase were checked in the laboratory.
Medium consist of 1 % starch showed maximum alpha amylase activity after 60 h of
fermentation. However manganese sulfate, zinc sulfate and EDTA showed inhibitory
effect on alpha amylase production by Bacillus sp. Haq et al., (2005a) reported the
choice of the appropriate surfactant for alpha amylase production by Bacillus subtilis
GCBM-25 during shake flasks. Various surfactants (laundry soap, detergent powder,
sulphonic acid, acyle benzene sulphonic acid, liquid soap, Tween 80, sodium silicate,
bath soap, sodium tripolyphosphate, sodium lauryl ether sulphate or sodium lauryl
sulphate) at rate 2.0 % (w/v) were screened for synthesis of enzyme. Among all the
surfactants, tested laundry soap proved to be superior with respect to alpha amylase
production (605 U/ml/min) after 44 h of fermentation using 4.0 % inoculum. The
enzyme production was enhanced (857 U/ml/min) with the addition of Millon soap at
rate 3.2 % (w/v) in the medium. However, addition of surfactants in the medium
reduced the thermostability from 70 to 50C.
Haq et al. (2005) reported the use of agricultural starchy substrate for alpha
amylase production by Bacillus licheniformis. The use of agriculture by products
made the medium economic. Soluble starch, hordium, pearl millet, rice, corn, gram
and wheat starch were screened for the alpha amylase production by parental and its
mutant derivative. The mutant strain B. licheniformis GCUCM-30 exhibited 10 fold
more enzyme production compared to parental strain when1.5 % pear millet and 0.25
% of nutrient broth was added to fermentation medium.

36
Anto et al. (2006) reported the alpha amylase production by B. cereus MTCC
1305 in solid state fermentation using wheat bran and rice flake manufacturing waste
as substrates. Maximum alpha amylase activity (942) U/g was obtained when wheat
bran was used as a substrate. Optimal conditions for alpha amylase production were
inoculum size 10 % substrate moisture ratio 1:1 and glucose, (0.04 g/g). Addition of
different nitrogen sources (0.02 g/g) showed decrease in enzyme production. Optimal
alpha amylase activity was observed at 55C and pH 5. Swain et al. (2006) reported
the alpha amylase production by B. subtilis isolated earlier from cow dung microflora.
The optimum temperature, pH and incubation period for amylase production were 5070C, 5-9 and 36 h, respectively. Enzyme secretion was very similar in the presence of
any of the carbon sources tested (soluble starch, potato starch, cassava starch, wheat
flour, glucose, fructose, etc.). Yeast extract and ammonium acetate (1 %) as nitrogen
sources gave higher yield compared to other nitrogen sources (peptone, malt extract,
casein, asparagine, glycine, beef extract) whereas ammonium chloride, ammonium
sulfate and urea inhibited the enzyme activity. Addition of Ca+2 (10-40 mM) to the
culture medium did not result in further improvement of enzyme production, whereas
the addition of surfactants (Tween 20, Tween 40, Tween 80, and sodium lauryl
sulphate) at 0.02 % resulted in 2-15 % increase in enzyme production. There were no
significant variations in enzyme yield between shake flask and laboratory fermenter
cultures. The purified enzyme was in two forms with molecular mass of 18.0 1 and
43.0 1 kDa in native SDS-PAGE.
Kathireasan and Manivannan (2006) isolated Penicillium fellutanum from
coastal mangrove soil and screened out the sound effects of different variables such

37
as pH, temperature, incubation time, salinity, carbon and nitrogen sources in shake
flasks fermentation for alpha amylase production. The fermentation medium with no
addition of seawater and supplemented with maltose and peptone as carbon and
nitrogen source was incubated for 96 h, at pH 6.5 and temperature 30C, gave
maximum alpha amylase production by P. fellutanum. Djekrif-Dakhmouche et al.
(2006) studied the alpha amylase production and optimization from A. niger ATCC
16404 .The statistical analysis has revealed that variation in agitation from 150 rpm 200 rpm has no effect on the alpha amylase production but it increased biomass. As
far as variation in pH from 5 to 6 has positive effect on alpha amylase production
while its effect on the biomass was negative. The addition of starch at 10 g/l to the
fermentation medium (an inductive substrate and carbon source) stimulated the alpha
amylase production, while it has no effect on biomass production. Calcium chloride at
1 g/l (a structural and stabilizing element for the alpha amylase) solely affect the
enzyme production. The use of other salts (manganese, iron sulfates as well as
magnesium chloride) seemed to be increased alpha amylase production but did not
effect either the production of protein or biomass.
Prakasham et al. (2007) reported fractional factorial design of Taguchi
methodology for the optimization of medium along with eight variables soluble
starch, corn steep liquor, casein, potassium dihydrogen phosphate, magnesium sulfate,
initial pH, incubation temperature and inoculum size for the amylase production in
submerged fermentation by A. awamori. Considerable enhancement approximately
48% in acid amylase synthesis was observed. The optimized fermentation medium
included in (%) soluble starch 3, CSL 0.5, KH2PO4 0.125, casein 1.5 at pH 4 and

38
31C. Shoji et al. (2007) reported a new submerged culture system of A. kawachii
NBRC4308 with the help of barley whose surface was wholly or partially covered
with husk. Both glucoamylase activity 150.8 U/ml and acid-stable alpha amylase
activity 7.7 U/ml were found in the supernatant in the presence of low concentration of
glucose.
Rao

et

al.

(2007)

investigated

the

formation

of

spores

from

B.

amyloliquefaciens B128 in shake flask cultivation. Fermentation media were

optimized by applying two steps approach. A rapid identification of an appropriate
carbon and nitrogen source was obtained by screening experimentation, and use of
response surface methodology (RSM). A five-level four-factor central composite
design was used to find out the highest spore yield at optimal level for lactose, tapioca,
ammonium sulfate and peptone. A noteworthy linear major effect was observed in the
case of topica and peptone, while lactose and ammonium sulfate produced no
important linear effect. Lactose-ammonium sulfate and lactose-peptone extensively
affected spore production. Optimum conditions for the alpha amylase production were
(g/l): lactose 12.7, tapioca 16.7, ammonium sulfate 1.8 and peptone 8. The predicted
spore production was 5.93 108 (no/ml). The real experimental results were in
concurrence with the prediction.
Suganuma et al. (2007) reported that highly humid climate of Japan facilitate the
growth of various molds. Among these molds A. oryzae was the most important and
popular in Japan, and has been used as yellow-koji in producing many traditional
fermented beverages and foods, such as Japanese sake, and soy sauce. The koji molds
black-koji and white-koji produce two types of alpha amylase, namely, acid-stable

39
(AA) and common neutral (NA).The latter enzyme was enzymatically genetically
similar to Taka amylase. In this review they investigated AA from three molds, A.
niger, A. kawachii and A. awamori, and the yeast Cryptococcus sp. regarding the
distinguishable properties between AA and NA. AA has many advantages in industrial
applications, such as its acid-stability, thermostability, and raw-starch digesting
properties. Bhanja et al. (2007) used Growtek bioreactor as modified solid state
fermenter to circumvent many of the problems associated with the conventional tray
reactors for solid state fermentation (SSF). A. oryzae IFO-30103 produced very high
levels of alpha amylase by modified solid state fermentation (mSSF) compared to SSF
carried out in enamel coated metallic trays utilizing wheat bran as substrate. High
alpha amylase yield of 15,833 U/ g dry solid in mSSF were obtained when the fungus
were cultivated at an initial pH of 6 at 32C for 54 h whereas alpha amylase
production in SSF reached its maximal (12,899 U g1 dry solid) at 30C after 66 h of
incubation. With the supplementation of 1 % NaNO3, the maximum activity obtained
was 19,665 U g1 dry solid (24% higher than control) in mSSF, whereas, in SSF
maximum activity was 15,480 U/ g dry solid in presence of 0.1 % Triton X-100 (20 %
higher than the control).
Tayeb et al. (2007) conducted the alpha amylase production using amplified
variants of B. subtilis (strain SCH) and of B. amyloliquefaciens (strain 267CH) in a
bioreactor with multiprotein-mineral media. The time course of fermentation in a
bioreactor revealed that the highest yield (about 8 x 104 U/ml within 6 h) by strain
SCH was obtained by applying: 3.5 % initial starch, 2 % additional starch after 19 h, 3
vvm aeration and 300 rpm agitation. The highest yield (about 19 x 104 U/ml within

40
100 h) by strain 267CH was obtained by applying: 2.5 % initial starch, 2 % additional
starch after 24 h, 3 vvm aeration, and 300 rpm agitation with the productivity after 60
h reaching only about 14 x 104 U/ml. Production occurred in both the logarithmic and
post logarithmic phases of growth. Maximum consumption of starch and protein
occurred during the first day of incubation. The optical density peak coincided with
enzyme production peak in case of strain SCH and preceded that of enzyme
production in case of strain 267CH. The alpha amylase produced by the two strains
was shown to be the liquefying and not both enzymes liquefied starch to a dextrose
equivalent of about 15-17 at 95C hence they are classified among thermostable alpha
amylases. They exhibited broad pH and temperature activity profiles. The optimum
pH for activity was 4-7 for alpha amylase produced by strain SCH and 4-8 for alpha
amylase produced by strain 267CH while the optimum temperatures for their activities
were in the range of 37 -75C at 0.5 % starch and in the range of 85 - 95C at 35 %
starch.
Poornima et al. (2008) isolated different strains of actinomycetes and tested
these strain for their ability to synthesize the alpha amylase. Among all the strains, the
strain AE-19 showed best alpha amylase production. This strain was identified as
Streptomyces aureofasciculus and selected for subsequent studies. The highest alpha
amylase production was obtained in the presence of mannose and L-histidine as
carbon and nitrogen source, 0.05 % sodium chloride at temperature 45C and pH 9.
These results indicated that strain can be successfully used for commercial alpha
amylase production after testing strain competence in large scale fermentations. Gupta
et al. (2008) studied the nutritional requirements of A. niger and the factors such as

41
incubation temperature, pH of medium, carbon and nitrogen sources, fermentaion
period, surfactants and concentration of metal ions. The experimental result showed
ideal carbon and nitrogen source for alpha amylase production was 0.5 % starch and
0.3 % peptone. The optimal pH, temperature and fermentation period were 5, 30C
and 5th day, respectively. Different surfactant at varying level such as Tween-80,
Triton X-100 and Sodium dodecyl sulphate at 0.02, 0.002 and 0.0002 % concentration,
respectively exhibited enhanced alpha amylase productivity. The major purpose of the
present study was to employ an appropriate fungal strain for extracellular alpha
amylase production and find out the fermentation period for the synthesis of alpha
amylase and to determine the effects of external substances that might increased the
synthesis of alpha amylase.
Esfahanibolandbalaie et al. (2008) reported the effect of many chemical and
physical factors on alpha amylase production by A. oryzae in shake flasks
fermentation via an Adlof-Kuhner orbital shaker. The impact of varying pH of
medium ranging from 4-7 was studied. The maximum alpha amylase production was
obtained at pH 6.2. Carbon and nitrogen source has discernible effect on the enzyme
production. The corn starch at level of 15 g/l proved to be best carbon source for alpha
amylase synthesis while glucose represses the alpha amylase production. The medium
consist of corn starch, sodium nitrate as inorganic nitrogen resulted in significant
enzyme production. Among the organic nitrogen sources yeast extract at the level of
2.5g /l was excellent nitrogen source. The impact of different temperatures and
agitation speed from 20 to 40C and 50 to 200 rpm, respectively was observed. The
maximum activity was obtained at 35C and 180 rpm. Planchot and Colonna (2008)

42
purified A. fumigatus (Aspergillus sp. K-27) extracellular alpha amylase to
homogeneity by using anion-exchange DEAE-cellulose and affinity -cyclodextrinSepharose chromatography. The purified enzyme was glycoprotein in nature which
was found to contain 15 % carbohydrate. The purified alpha amylase exhibited an
isoelectric point of 3.7, and SDS PAGE estimated that purified enzyme possess a
molecular weight of 65,000. A large

number of neutral hydrophobic residues were

present in an amino acid. The optimum enzyme activity was obtained at pH 5.5, and
the enzyme showed stability at 40C. It hydrolyzed amylose and amylopectin, with
respective Km of 0.42 and 7.7 mg mL- 1 and kcat/K m of 3.4 and 2.5 mL mg

-1

min-1.

The main end-products of maltohexaose, hydrolysis were glucose and maltose. While
intermediate products were maltotriose, maltotetraose, and maltopentaose having an anomeric configuration. Although its capability to gradually degrade some 1-6
linkages, purified enzyme ought to be classified as an alpha-amylase.
Leman et al. (2009) reported alpha amylase from A. oryzae had only very little
effect on the side chain segments of the amylopectin molecules and the reason might
be enzyme hydrolysis the segments of internal chain. Singh et al., (2009) investigated
the effect of various agricultural by products as a substrate such as wheat bran, wheat
straw, rye, straw on the alpha amylase production by Humicola lanuginose in solid
state fermentation. Wheat bran proved to be good substrates for starch degrading
enzymes because highest alpha amylase production was observed when wheat bran
was used as a substrate. Various variables such as moisture content, incubation time
inoculum size and carbon source has marked effect on the enzyme production. It was
noted the optimum condition for the alpha amylase production by Humicola

43
lanuginose in SSF was incubation period 144 h, initial moisture content 90 %, initial
pH of medium 6, incubation temperature 50C , size of inoculum 20 % and soluble
starch as best carbon source.
Shafique et al. (2009) tested the five strains of fungi belonging to two
filamentous fungi A. niger and A. flavus for their ability to produce alpha-amylase.
The different chosen strains were cultivated on two different typed of media i.e.,
potato dextrose agar (PDA) and enzyme production medium (EPM), the pH of
medium was fixed at 3 level i.e., 4.5, 5.5 and 6.5. The efficiency or ability of strains
was estimated on the basis of the formation of hydrolysis zone. EPM medium at pH
4.5 was best for the highest activity of alpha amylase. Strain 74 and strain 198 of A.
niger and strain 209 and strain 231 of A. flavus gave best result on solid media; so
these strains were selected for the alpha amylase production in submerged
fermentation. All the selected strains showed highest activity of alpha amylase after 48
h in shake flasks.

44

Uses of alpha amylase

Starch is a major storage product of many economically important crops such as
wheat, rice, maize, tapioca, and potato. A large-scale starch processing industry has
emerged in the last century. In the past decades, we have seen a shift from the acid
hydrolysis of starch to the use of starch-converting enzymes in the production of
maltodextrin, modified starches, or glucose and fructose syrups. Currently, these
enzymes comprise about 30 % of the world's enzyme production. Besides the use in
starch hydrolysis, starch-converting enzymes are also used in a number of other
industrial applications, such as laundry and porcelain detergents or as anti-staling
agents in baking. A number of these starch-converting enzymes belong to a single
family: the alpha amylase family or family13 glycosyl hydrolases. This group of
enzymes share a number of common characteristics such as a (/)8 barrel structure,
the hydrolysis or formation of glycosidic bonds in the conformation, and a number
of conserved amino acid residues in the active site. As many as 21 different reaction
and product specificities are found in this family.

Bread and chapatti industry

The quantities, taste, aroma and porosity of the bread are improved by using the enzyme
in the flour. More than 70 % bread in U.S.A, Russia and European countries contain
alpha amylase. Amylases play important role in bakery products (Goodwin and Mercer,
1972). For decades, enzymes such as malt and fungal alpha-amylases have been used in
bread-making. The significance of enzymes is likely to raise as consumers insist more
natural products free of chemical additives. For example, enzymes can be employed to

45
replace potassium bromate, a chemical additive that has been prohibited in a number of
countries. The dough for bread, rolls, buns and similar products consists of flour, water,
yeast, salt and possibly other ingredients such as sugar and fat. Flour consists of gluten,
starch, non-starch polysaccharides, lipids and trace amounts of minerals. As soon as the
dough is made, the yeast starts to work on the fermentable sugars, transforming them
into alcohol and carbon dioxide, which makes the dough rise. The major component of
wheat flour is starch. Amylases can degrade starch and produce small dextrins for the
yeast to act upon. The alpha-amylases degrade the damaged starch in wheat flour into
small dextrins, which allows yeast to work continuously during dough fermentation,
proofing and the early stage of baking. The result is improved bread volume and crumb
texture. In addition, the small oligosaccharides and sugars such as glucose and maltose
produced by these enzymes enhance the Maillard reactions responsible for the browning
of the crust and the development of an attractive baked flavour (Lundkvist et al., 2007).

Textile industry
Textile industries are extensively using alpha amylases to hydrolyze and solubilize the
starch, which then wash out of the cloth for increasing the stiffness of the finished
products. Fabrics are sized with starch. Alpha amylase is used as desizing agent for
removing starch from the grey cloth before its further processing in bleaching and
dyeing. Many garments especially the ubiquitions Jean are desized after mashing.
The desired fabrics are finally laundered and rinsed (Iqbal et al., 1997; Allan et al.,
1997).

46

Sugar and Glucose industries

Alpha amylase plays a very important role in the production of starch
conversion products of low fermentability. The presence of starch and other
polysaccharides in sugar cane creates problem throughout the sugar manufacturing
which is minimized or eliminated by the action of alpha amylase. The high quality
products depends upon the efficiency of the enzyme which lead to low production,
costs for the starch processor has increased (De-cordt et al., 1994; Ensari et al., 1996;
Hamilton et al., 1998). Many industries used alpha amylases for the production of
glucose. Enzyme hydrolyzed the starch and converted it into glucose. They hydrolyze
-1,4 glucosidic linkage in the starch polymer in a random manner to yield glucose
and maltose (Akiba et al., 1998). Therefore, alpha amylase is extensively used in
many industries for the production of glucose (Shetty and Crab, 1990). This process
also gives the water-soluble dextrin.

Alcohol Industry
Alpha amylases convert starch in to fermentable sugars. Starches such as grain;
potatoes etc. are used as a raw material that helps to manufacture ethyl alcohol. In the
presence of amylases, the starch is first converted in to fermentable sugars. The use of
bacterial enzyme partly replaces malt in brewing industry, thus making the process
more economically significant. Alpha amylase can also carries out the reactions of
alcoholysis by using methanol as a substrate (Santamaria et al., 1999).

47

Paper industry
Starch paste when used as a mounting adhesive modified with additives such as
protein glue or alum, frequently, causes damage to paper as a result of its
embrittlement. Starch digesting enzymes, e.g. alpha amylase, in immersion or as a gel
poultice are applied to facilitate its removal. Alpha amylase hydrolyzed the raw starch
that is used for sizing and coating the paper instead of expensive chemically modified
starches. So, starch is extensively used for some paper size press publications (Okolo
et al., 1996).

Detergent, Building product and Feed industries

In detergent industries, the enzyme alpha amylase plays a vital role. It is widely used
for improvement of detergency of laundry bleach composition and bleaching with out
color darkening (Borchet et al., 1995; Atsushi and Eiichi, 1998). The addition of
enzyme stabilizes the bleach agent and preserves effectiveness of the bleach in laundry
detergent bar composition (Onzales, 1997; Mirasol et al., 1997) Modified starch is
used in the manufacture of gypsum board for dry wall construction. Enzyme modified
the starch for the industry use. Many starches or barely material are present in the
feed. So, the nutritional value of the feed can be improved by the addition of alpha
amylase.

Chocolate industry
Amylases are treated with cocoa slurries to produce chocolate syrup, in which
chocolate starch is dextrinizing and thus syrup does not become thick. Cocoa flavored
syrups having a high cocoa content and excellent stability and flow properties at room

48
temperature may be produced by using an amylolytic enzyme and a sufficient
proportion of Dutch process cocoa to provide a syrup pH of 5.5 to 7.5. The syrup is
made by alternate addition of cocoa and sweetener to sufficient water to achieve a
solids content of about 58 to 65 weight percent, adding an amylolytic enzyme, heating
to a temperature of about 175 -185F for at least 10 to 15 min, raising the temperature
to about 200 F. and cooling. The stabilized cocoa flavored syrups may be added at
room temperature to conventional non-acid confection mixes for use in the production
of quiescently frozen chocolate flavored confections (Ismail et al., 1992)