Review of Literature

2.0

REVIEW OF LITERATURE
Amylases are starch degrading enzymes. They are widely distributed

in microbial, plant and animal kingdoms (Banks et al, 1975). They degrade
starch and related polymers to yield products characteristic of individual
amylolytic enzymes. Initially the term amylase was used originally to
designate enzymes capable of hydrolysing -1, 4- glycosidic bonds of
amylose, amylopectin, glycogen and their degradation products (Damien et
al, 2010). They act by hydrolysing bonds between adjacent glucose units,
yielding products characteristic of the particular enzyme involved (Dhanya
et al, 2009).
In recent years a number of new enzymes associated with degradation
of starch and related polysaccharides structures have been detected and
studied (Mohammad et al, 2010). The enzymes having potential commercial
importance of microbial origin that split -1,4 or -1,4 and/or -1,6 bonds
in these structures, may be divided in the following six classes.
1. Enzymes that hydrolyse alpha-1,4 bonds and bypass alpha -1,6
linkages e.g. -amylase (endo acting amylases).
2. Enzymes that hydrolyse -1,4 and cannot bypass -1,6 linkages
e.g. -amylase (exoacting amylases producing maltose as a major
end product).
3. Enzymes that hydrolyse -1,4 and -1,6 linkages e.g. amylo
glucosidase (gluco amylase) and exo acting amylase.
4. Enzymes that hydrolyse only -1,6 linkages e.g. pullulanase and
other debranching enzymes.
5. Enzymes that hydrolyse preferentially -1,4 linkages in short chain
oligosaccharides produced by the action of other enzymes on
amylose and amylopectin e.g. -glucosidases.

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6. Enzymes that hydrolyse starch to a series of non reducing cyclic
D-glucosyl polymers called cyclodextrins or sachardinger dextrins
e.g. Bacillus macerans amylase (cyclodextrin producing enzyme)
(Archana et al, 2011)
2.1

STARCH
Before describing the action pattern and properties of amylolytic

enzymes it is essential to discuss the features of the natural substrate,

starch is a major reserve carbohydrate of all higher plants (Encarnacion et
al, 2011). In some cases it accounts for as high as 70% of the undried plant
material. It occurs in the form of water insoluble granules (Guadalupe et al,
2011). The size and shape of the granules is often characteristic of the plant
species from which they are extracted. When heated in water the hydrogen
bonds holding the granules together begin to weaken and this permits them
to swell and gelatinize (Tomasz et al , 2011).Ultimately they form paste or
dispersion, depending on the concentration of polysaccharide (Xuerong et al,
2008). Starches are produced commercially from the seeds of plants, such
as corn, wheat, sorghum or rice, from the tubers and roots of the plants
such as cassava, potato, arrowroot and the pith of sago palm (Cynthia et al,
2011).The major commercial source of starch is corn from which it is
extracted by a wet milling process (Dhanya et al, 2009).
Starch is a heterogeneous polysaccharide composed of two high
molecular weight entities called amylose and amylopectin (Damien et al,
2010). These two polymers have different structures and physical properties.

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TABLE 2.1.1 COMPARISON OF AMYLOSE AND AMYLOPECTIN
Properties

Amylose

Amylopectin

Basic structure

Linear

Branched

Stability in aqueous solution

Retrogrades

Stable

Degree of Polymerization

C.103

C.104 - C.105

Average chain length

C.103

C.20-25

Beta amylase hydrolysis

87%

54%

Beta amylase & Debranching enzyme 98%

79%

hydrolysis
Maximum Iodine complex

650 nm

550 nm

Starch may be separated into two components by addition of a polar

solvent, e.g. n-butanol, to a dispersion of starch (Natasa et al, 2011). The
insoluble amylose complex can then be separated from soluble amylopectin
fraction. Amylose is composed of linear chains of -1,4 linked D-glucose
residues(Chi-Wenlin et al, 2011). Hence it is extensively degraded by amylase. Some amylose is not totally degraded to maltose by this enzyme.
Amylose has a degree of polymerization of several thousands of glucose
units (Bank et al 1975; Maryam et al, 2010). Because of the molecular
shape and structure of amylase, it is not stable in aqueous solution and
retrogrades (precipitates spontaneously), (Ahmad et al, 2010). This is
because linear chains align themselves by hydrogen bonding and thus forms
aggregates. This process is irreversible retrograded amylase will only
dissolve in alkaline solution (Burhan et al, 2008). Amylose has considerable
viscosity in alkaline solutions due to its molecular shape. Amylase this
forms complex with iodine to form intense blue colour and this forms the
basis of a method for quantitative determination of amylase (Aw et al, 1969).
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Amylopectin may account for 75 to 80% of most starches. It has
molecular weight in excess on 10-10 and has a branched structure
composed

of

chains

about

20-25

-1,4

linked

D-glucose

residues.

Amylopectin which is branched by -1,6 D-glucosidic bonds (Henrissat et al,

1991).In aqueous solutions, amylopectins are relatively stable due to
branched molecules and are not able to form compact aggregates. There is
no apparent relationship between the limiting viscosity number and the
degree of polymerization. Due to the nature of branched structure, the
iodine binding power is reduced (Aw et al, 1969). The branched components
of starch are amylopectin which has different types of chains referred to as
A, B and C chains (Mohammad et al, 2010).
The hydrolysis of starch may be carried out using either acid or
enzyme as catalyst (Diana et al, 2007). Enzyme hydrolysis has several
advantages; it is more specific, therefore fewer by products are formed, and
hence yields are higher. Conditions for enzyme hydrolysis are milder
therefore refining stages to remove ash and colour is minimized. The
enzymatic hydrolysis of starchy has been practiced on an industrial scale for
many years and is gradually replacing the traditional acid hydrolysis
process (Kazunari et al, 2011).
2.2

SOURCES OF ALPHA AMYLASE

Alpha amylases are ubiquitous enzymes produced by plants, animals

and microbes, where they play a dominant role in carbohydrate metabolism

(Varel et al, 1994). Amylases from plant and microbial sources are employed
for centuries as food additives (Mabel et al, 2008). Barley amylases are used
in Brewing industry. Fungal amylases are widely used in preparation of
oriental foods (Popovic et al, 2009). Fungal and bacterial amylases are
mainly used for industrial applications due to their cost effectiveness,
consistency, less time and space requirement for production and ease of
process optimization and modification (Ellaiah et al, 2002).

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Among bacteria Bacillus.sp is widely used for the production of
amylases. Species like B.subtilis, B.stearothermophilus, B.licheniformis,
and B.amyloliquefaciens are known to be good producers of alpha amylase
(Harshemi et al, 2011). Similarly filamentous fungi have been widely used
for the production of amylases for centuries (Juliana et al, 2011). As these
moulds are known to be prolific producers of extracellular proteins, they are
widely exploited for the production of different enzymes including alpha
amylases (Kozunari et al, 2011). Fungi belonging to the genus Aspergillus
have been most commonly employed for the production of alpha amylase.
Production of enzymes by solid state fermentation using these moulds
turned a cost effective production technique (Parveen et al, 2011).
2.3

FERMENTATIVE PRODUCTION OF AMYLASE

To meet the demand of industries, low cost medium is required for

the production of alpha-amylase (Aliyu et al, 2011). Both SSF and

submerged fermentation (SmF) could be used for the production of amylase,
although traditionally these have been obtained from submerged cultures
because of easy handling and greater control of environmental factors such
as temperature and pH (Xusheng et al, 2011).Mostly synthetic media have
been used for the production of bacterial amylase through SmF (Ajay et al,
2010). The contents of synthetic media such as nutrient broth, soluble
starch, as well as other components are very expensive and these could be
replaced with cheaper agricultural by products for the reduction of the cost
and the medium(Solange et al, 2010). The solid substrate may provide only
support and nutrition (Hashemi et al, 2011). SSF is considered as an
interesting alternative since the metabolites so produced are concentrated
and purification is of less quality (Nasrin et al, 2010). SSF is preferred to
SmF because of simple technique, low capital investment, lower levels of
catabolite repression and end product inhibition, low waste water out put
better product recovery and high quality has been reported to produce
promising results (Ajay et al, 2010) other substrates such as sunflower
meal, rice husk, cotton seed meal, soybean meal, rice husk, cotton seed
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meal, soy bean meal, and pearl millet and rice bran have been tried for SSF
(Maryam et al, 2010).
SSF technique is generally confined to the process involving fungi
(Kiran et al, 2010). However, successful bacterial growth in SSF is known
much in natural fermentation (Lonsane et al, 1990). The production of alpha
amylase by SSF is limited to the genus Bacillus like B.subtilis, B.polymaxa,
B.mesentiricus, B.vulgarus, B.coagulans, B.megaterium and B.licheniformis
have been used for alpha amylase production in SSF (Natasa et al,
2011).The production of bacterial amylase using alpha amylase technique
requires less fermentation time which leads to considerable reduction in the
capital and recurring expenditure (Li Zhuang et al, 2011). Research on the
selection of suitable substrates for SSF has mainly been centred around
agro industrial residues due to their potential advantages for filamentous
fungi which are capable of penetrating into the hardest of these solid
substrates, aided by the presence of turgor pressure at the tip of the
mycelium (Maryam et al, 2010). In addition, the utilization of these agro
industrial wastes, not only provides alternative substrates but also on the
other hand helps in solving pollution problems (Priya et al, 2011). Table 1
summarizes various agro residues reported for microbial alpha amylase
production.
TABLE 2.3.1 VARIOUS SUBSTRATES USED FOR ALPHA AMYLASE
PRODUCTION
Substrate

Organism

Activity (U/g)

Wheat bran

Bacillus sp.PS-7

464,000

Spent brewing grain

A.oryzae NRRL 6270

6583

Maize bran

B.coagulans

22956

Rice bran

Bacillus sp.PS-7

145,000

Coconut oil cake

A.oryzae

3388

Amaranthus grains

A.flavus

1920

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2.4

PROCESS OPTIMIZATION
Optimization of the various parameters and manipulations of media

are one of the most important techniques used for the over production of
amylase in large quantities (Balasubramanien et al, 2011). To meet
industrial demands production of alpha amylase in fungi is known to
depend on both morphological and metabolic state of the culture (Juliana et
al, 2011). Growth of mycelium is crucial for extracellular enzyme like alpha
amylase (Sangeeta et al, 2009). Various physical and chemical factors have
been known to effect the production of alpha amylase such as temperature,
pH, Incubation period, carbon, nitrogen sources, surfactants, phosphate,
different metal ions, moisture and agitation with respect to SSF and SmF
(Ellaiah et al, 2002).
2.4.1

TEMPERATURE:
The influence of temperature on amylase production is related to the

growth of the organism (Pandey et al, 1990). Hence the optimum

temperature depends on whether the culture is mesophilic or thermophilic.
Among the fungi most amylase production studies have been done with
mesophilic fungi within the temperature range of 25-37oC (Takahiro et al,
2011). A raw starch degrading amylase was produced by Aspergillus ficum
at 30oC by Hayashida et al in 1986. Yeast such as Saccharomyces kluyveri
and S.cerevisiae was reported to produce alpha amylase at 30oC (Moller et al
2004). Amylase production at optimum level has been reported between 5055oC for the thermophilic fungal cultures such as Talaromyces emersonni,
Thermomonospora fusca etc (Ahmad et al, 2010).
2.4.2

pH:
pH is one of the important factors that determine the growth and

morphology of microorganisms as they are sensitive to the concentration of

hydrogen ions present in the medium(Ellaiah et al, 2002). Earlier studies
have revealed that fungi required slightly acidic pH and bacteria required
neutral pH for optimum growth. pH is known to effect the synthesis and
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secretion of alpha amylase just like its stability (Yakup et al, 2010). Fungi of
Aspergillus sp. such as A.oryzae, A.ficuum and A.niger were found to give
significant yields of alpha amylase at pH equal to 5.0 to 6.0 in SmF (Parveen
et al, 2011). Alpha amylase producing yeast strains such as S.cerevisiae and
S.kluyveri exhibited maximum enzyme production at pH 5.0 (Samrat et al,
2011).
2.4.3

CARBON SOURCES:
Carbon sources such as galactose, glycogen and Inulin have been

reported as suitable substrates for the production of amylases by

B.licheniformis and Bacillus.sp.1-3 (Xusheng et al, 2011). Starch and
glycerol were known to increase enzyme production in B.subtilis IMG22,
Bacillus sp, PS-7 and Bacillus sp.1-3(Maryam et al, 2010). Soluble starch
has been found as the best substrate for the production of alpha amylase by
B.stearothermophilus (Srivastava et al 1986). Bacillus sp. was noted to give
a maximum raw starch digesting amylase in a medium containing lactose
(1%) and yeast extract (15%). Thermomyces lanuginosus was reported to
give maximum alpha amylase yield when maltodextrin was supplemented to
the medium (Nguyen et al, 2000). Agricultural wastes are being used for
both liquid and solid fermentation to reduce the cost of fermentation media.
The waste consists of carbon and nitrogen sources necessary for the growth
and metabolism of organism. These nutrients sources include orange waste,
peer millet starch, potato, corn, tapioca, wheat and rice as flours (Lin Hui et
al, 2011).
2.4.4

NITROGEN SOURCES:
Soybean meal was found as the best nitrogen source for alpha

amylase by Bacillus sp. 1-3. Tanyildizi et al reported that peptone increased

enzyme activity while yeast extract exhibited no effect on alpha amylase
production (Arpana et al, 2011). Strains of Bacillus stearothermophilus and
B.amylolyticus

secreted

maximum

alpha

amylase

in

medium

supplemented with 1% peptone, 0.5% yeast extract and 0.5% maltose under
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vigorous shaking conditions (Elif et al, 2005), compared the influence of
organic and inorganic nitrogen sources and reported peptone to be a better
nitrogen source for enzyme production by B.licheniformis SPT 278 than
ammonium phosphate, the best among inorganic nitrogen sources. Lasparagine was reported to be one of the most promising nitrogen sources
for alpha amylase production by Thermomyces lanuginosus (Adinarayana et
al, 2005). Yeast extract also resulted in a significant alpha amylase yield.
Supplementation of Casein hydrolysate to the medium resulted in 143%
increase in alpha amylase productivity by A.oryzae 1560 compared to
ammonia.
2.4.5

SURFACTANTS:
Surfactants in the fermentation medium are known to increase the

secretion of proteins by increasing cell membrane permeability. Therefore

addition of these surfactants is used for the production of extracellular
enzymes (Samrat et al, 2011). Addition of tween 80 (1.3%) to the
fermentation medium increased alpha amylase production by 2 fold in
Thermomyces lanuginosus (Sivaramakrishnan et al, 2006). A study on the
effect of supplementation of Poly ethylene glycols (PEG) (molecular mass of
600,3000,4000,8000 and 20,000) in fermentation medium for alpha
amylase production by two bacillus sp. Researchers indicated that 5% PEGs
600 and PEG 3000 yielded 31% increase in enzyme production by
B.amyloliquefaciens and 21% increase by B.subtilis (Goes et al, 1999).
2.4.6

METAL IONS:
Supplementation of salts of certain metal ions provided good growth

of microorganisms and thereby better enzyme production as most alpha

amylases are known to be metalloenzymes (Zoe et al, 2006). Ca+2 are
reported to be present in majority of these enzymes. Addition of Calcium
chloride to the fermentation media increased the enzyme production (Arthur
et al, 1996). Positive results of the influence of CaCl2 (0.1%) and NaCl (0.1%)
on alpha amylase production in SSF using Amaranthus grains as substrates
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were recorded (Vishwanathan et al, 2001). LiSo4 (25 mM) and MgSo4 (1mM)
increased alpha amylase production by Bacillus sp.1-3 (Reeta et al, 2009)
but FeCl3 and MgSo4 exhibited negative influence on alpha amylase
production ( Vishwanathan et al 2001).
2.4.7

MOISTURE CONTENT:
Moisture is one of the most important parameters in SSF that

influences the growth of the organism and thereby enzyme production

(Pandey et al, 2000). Low and high moisture levels of the substrate effect the
growth of the microorganisms resulting in lower enzyme production (Ellaiah
et al, 2002). High moisture content leads to reduction in substrate porosity,
changes in the structure of substrate particles and reduction of gas volume.
Bacteria are generally known to require initial moisture of 70-80%. Alpha
amylase production by Bacillus licheniformis M27 was highest with 65%
initial moisture content in an SSF system (Namita et al, 2007). Significant
decrease in enzyme production was observed with high increase in moisture
content which was due to the decrease in the rate of oxygen transfer.
Studies indicated that enzymes titres could be increased significantly by
agitation of the medium with high moisture content (Lonsane et al, 1990).A
thermotolerant B.subtilis requires initial moisture of 30% for its growth and
maximum enzyme production (Ahmad et al, 2010).
2.4.8

PARTICLE SIZE OF THE SUBSTRATE:

In SSF, particle size of the substrate effects growth of the organism

and thereby influences the enzyme production (Ellaiah et al, 2002). The
adherence and penetration of micro organisms as well as the enzyme action
on the substrate clearly depend upon the physical properties of the
substrate such as the crystalline or amorphous nature, the accessible area,
surface area, porosity, particle size etc (Chen et al, 2011). In all the above
parameters, particle size plays a major role because all these factors depend
on it (Pandey et al, 1991). Smaller substrate particles have greater substrate
surface area for growth but inter particle porosity is lower (Pandey et al,
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1991).For larger particle sizes, the porosity is greater but the saturated
surface area is smaller hence determination of particle size corresponding to
optimum growth and enzyme production is necessary (Reeta et al, 2009).
2.5

PURIFICATION:
Down stream processing for the production of pure enzymes can

generally constitute a major percentage of overall production cost especially

if end purity requirements are stringent (Lalit et al, 2010). Purification
process in down stream processing after fermentation strongly depend on
the market, processing cost, final quality and available technology. Most
enzymes are purified by Chromatographic techniques after crude isolation
by precipitation and membrane separations (Prakash et al, 2009). The need
for large scale cost effective purification of proteins has resulted in evolution
of techniques that provide fast, efficient and economical protocols in fewer
processing steps (San-Lang et al, 2011). Purification techniques that
produce homogenous preparation of amylase in a single step are given in
below table (Anni linden et al, 2000).

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2.5.1 METHODS OF ONE STEP PURIFICATION OF ALPHA AMYLASES
Method

Adsorbent

Affinity Adsorption

Beta

Chromatography

cyclodextrin-

Yield

Purification Reference

(%)

fold

95

Liao et al

69

51

Amritkar

iminodiacetic
acid- Cu+2
Expanded bed

Alginic acid

chromatography

cellulose cell

et al

beads
High speed counter

PEG 4000

current

aqueous two

chromatography

phase system

Magnetic affinity

Magnetic

adsorption

alginate

73.1

Zhi et al

88

Safari kova
et al

microparticles
Substitute affinity

Insoluble corn

method

starch at 4oC

2.6

78

163

Najafi et al

IMPROVEMENT OF THE STRAIN

The mutant strains of Bacillus have better ability to produce alpha

amylase, which can be derived by mutagenesis and extra screening. Both

chemical mutagenic agents as well as UV irradiations can be used to
improve the Bacillus strains for the production of alpha amylase. The UV
irradiations produce mutants by the photolysis of pyramidines to from
dimers. These dimers can cause errors in the replication, which result in
mutation (Guadalupe et al, 2011). During the course of ultra violet studies
with the bacillus subtilis (ATCC6051), a mutant was obtained which was
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stimulated by a factor in yeast extract with glucose inorganic salts medium
containing monoacids, vitamins, purines and a pyramidine. This mutant
was a stable one and can be stored at 5oC for several months.
Markkanen and suihko (1947) found that UV irradiation was suitable
mutagen of alpha amylase and proyteolytic enzyme production by Bacillus
subtilis. Cells exposed to UV radiation produced large number of
permutations, mostly as a result of dimerization of thiamine. Those
premutants usually under go partial or complete repair on return to visible
light, when a specific enzyme acts to separate the dimerized thiamine
molecules. Irradiation causing death rate of 90% was found to be most
effective in the production of mutants with improved amylase yields. Among
mutants, the best frequencies of positive mutations were 1:50 and 1:20 for
amylase and proteolytic enzyme, respectively.
Bailey et al, (1979) employed various mutagenic agents in
succession to produce mutant strains of Bacillus subtilis with improved
yield of amylase. The parent strain had previously been selected as a good
producer of amylase. Highly productive mutant strains were selected
through out the work as the basis for further treatment. In shake flask
cultures, the yields of amylase of the best strains were double that of parent
strain and in Fermentor cultivation the improvement was even greater.
Yu et al (1986) have used NTG as mutagen for the mutation of
Bacillus subtilis. Two mutant strains K1 and K5 were isolated from 2500
isolates. These strains gave increase in the yield of enzyme than the parental
strain BF7658. The average yield of alpha amylase of K1 and K5 strains
were 320 and 381 U/ml. respectively.
Shah et al (1989) have isolated a high yielding mutant of B.subtilis
by subjecting its parental strain and subsequent highest yielding mutant,
after each exposure, to successive to N-Methyl-N-Nitro-N-Nitrosoguanidine
(NTG) at various concentrations and finally to a single UV irradiation. This
mutant secretes 5-fold more alpha amylase activity than the parental strain.
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In screening higher producer of thermostable alpha amylase, Bacillus
licheniformis B198 was chosen as an original strain, which was treated
repeatedly with various mutagens. The mutant A.4041 was selected after
repeated natural selection. The thermostable alpha amylase activity of this
mutant was in 100 fold of the original one, reaching 200U/ml in the shake
flask. The mutant was resistant to catabolite repression by glucose (Xuezhi
et al, 1991).
Qirang and Zhao (1994) investigated the selection and breeding of a
high productivity of a amylase from multi resistant mutant of Bacillus. Jin
et al ,(1998) have developed a hyper producing alpha amylase mutant of
Bacillus licheniformis. The mutant shows 50 times higher enzyme than the
parental strain.
Bin et al (1999) screened out alpha amylase high producing strains
from Bacillus subtilis. In screening high producer of alpha amylase, Bacillus
subtilis 14140 was chosen as an original strain. The strain was treated
repeatedly with N-Methyl-N-Nitro-N-Nitrosoguanidine (NTG). After screening,
the mutant B.subtilis GS was selected. The enzymatic activity of alpha
amylase was raised from 3000U/ml. Effect of carbon sources and nitrogen
sources on the formation of alpha amylase were also studied in the shake
flask. Niziolek, (1998) investigated the production of extracellular, amylolytic
enzymes in 41 strains of the genus Bacillus representing 13 species using
different liquid media and cultivation temperature of 30oC and 38oC. It was
found that 8 strains were amylase negative, 19 strains were low productive
and 12 were medium productive strains (10-25 U/ml). B.subtilis AS-1-108,
B.subtilis NCIB 8159 and B.licheniformis NCIB 7198 strains were included
among the higher producers as they produced about 370, 170 and 40 U/ml
of alpha amylase. The amylase production by B.subtilis was variously
affected by medium composition and temperature of cultivation. The
enzymes from B.subtilis AS-1-108 and NCIB 8159 strains were more thermo
sensitive than those of the medium productive strains of B.subtilis. The

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action pattern of alpha amylase from B.subtilis strains was affected by pH
and temperature.
Alkaline amylase producing mutants were successfully induced from
this strain by mutagenesis with two different agents like ultra violet light
and NTG (1-methyl 1-3-nitro-nitroguanidine).In the first mutation step (UV
mutagenesis) they selected a mutant strain, Bacillus sp. ICCF 276/18 which
was induced with UV mutant strain. The amylolytic activity of Bacillus sp.
ICCF 276/18 in the optimized medium was 17.5 U/ml, being approximately
2.5 higher fold than that of the wild strain (Dinu et al, 2001).
Haq et al (2002) investigated the biosynthesis of alpha amylase by
chemically treated mutant of B.subtilis GCBUCM-25.The strain of B.subtilis
was treated with NTG for different intervals of time (5-60 min). One hundred
mutant strains were isolated and tested for the production of alpha amylase
and Bacillus subtilis GCBUCM-25 gave maximum production of enzyme
(2210 U/ml). The optimum conditions for the production of alpha amylase
were sodium nitrate as nitrogen source, pH 7.5 phosphate buffer and 4mM
CaCl2 as diluents.
Table 2.6.1 Alpha Amylases Exhibiting Different Temperature Stability
Residual
activity

Optimum
Reference
Temperature

Lactobacillus
50-60
manihotivarans

70 (50oC for
1.0h)

55

Aguilar et al

Bacillus sp 1-3

65-100

50 (80oC for
2.5h)

70

Goyal et al

Pyrococcus
furiosus

80-100

50 (98oC for
13h)

100

Viellie et al

Aspergillus
tamarii

50-60

90 (65oC for
3h)

55

Moreria et al

Cryptococcus
flavus

50-60

60 (60oC for
60 min)

50

Wanderley et
al

Organism

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2.6.2

CONCENTRATION EFFECT OF DENATURING AGENTS ON ALPHA

AMYLASE ACTIVITY

Inhibitor

Concentration effect

Organism

Reference

EDTA

10mM Resistant

Bacillus sp. L1711

Bernharsboteer

10mM Inhibitory

Bacillus sp.1-3

Goyal et al

1% slight inhibition

B.halodurmas

Hagihara et al

SDS

LBK 34
Urea

8M Inhibitory

Bacillus sp. ANT-6

Burhan et al

Hydrogen

1.8M Resistant

Bacillus KSM-K38

Hagihara et al

Peroxide

2.6.3

Applications Of Amylases In Various Industrial Sectors

Sector
Food Industry

Applications
Production of glucose syrup,
crystalline glucose

References
Hans et al,
2009.

Production of HFCS
Production of Maltose syrups
Reduction of viscosity of sugar syrups
Reduction of haze formation in juices
Solubilization & Saccharification of
starch for alcohol fermentation in
brewing industries
Retardation of staling in baking
industry
Detergent
Industry
Murali Krishna. CH

Used as an additive to remove starch based

dirts

Gupta et al,
2005
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Review of Literature
Paper Industry

Reduction of viscosity of starch for

appropriate coating of paper

Ellaiah et al,
2002

Textile
Industry

Warp sizing of textile fibres

Reeta et al,
2009

Pharmaceutical Used as a digestive aid

industry

2.7

Guler et al,
2010

APPLICATIONS OF AMYLASE
The history of the industrial production of enzymes dates back to the

time when Dr. Jhokichi Takamine began the production of digestive enzyme
preparation by wheat brankoji culture of Aspergillus oryzae in 1894.
Industrial production of dextrose powder and dextrose crystals from starch
using -amylase and glucoamylase began in 1959 (Pandey et al, 2000).
Since then, amylases are being used for various purposes. Conversion of
starch into sugar, syrups and dextrins forms the major part of the starch
processing industry (Noda et al, 2001). The hydrolysates are used as carbon
sources in fermentation as well as sources of sweetness in a range of
manufactured food products and beverages. Hydrolysis of starch to products
containing glucose, maltose etc, is brought about by controlled degradation
(Hans et al, 2009; Uma et al, 2007).Some of the applications of amylase are
as follows
2.7.1 LIQUEFACTION
Liquefaction is a process of dispersion of insoluble starch granules
in aqueous solution followed by partial hydrolysis using thermostable
amylases. In industrial processes, the starch suspension for liquefaction is
generally in excess of 35% (w/v) (Damien et al, 2010). Therefore the viscosity
is extremely high following gelatinization (Vander et al, 2002). Thermostable
-amylase is used as a thinning agent, which brings about reduction in
viscosity and partial hydrolysis of starch. Retrogradation of starch is thus
avoided during subsequent cooling (Sang-Lang et al, 2000).
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The traditional thinning agent used in starch technology was acid
(hydrochloric or oxalic acids). The introduction of thermostable -amylases
has meant milder processing conditions. The formation of by products is
reduced and refining and recovery costs are lowed (Dhanya et al, 2009).
In the enzymatic process the hydrolytic action is terminated when
the average degree of polymerization is about 10-12. Two distinct types of
thermostable -amylases are commercially available and used extensively in
starch processing technology (Tomasz et al, 2011). The amylase of Bacillus
amyloliquefaciens was the first liquefying -amylase used on a large scale.
Later a more heat stable enzyme from Bacillus licheniformis was introduced
commercially (Madsen et al, 1973). Liquefaction can be done by two
methods like:
Single stage enzyme liquefaction: In 1973, Novo industry at
Copenhagen developed and patented the process. In this process, starch
slurry containing 30-40% dry solids is prepared in the feed tank. The PH is
adjusted to about 6-6.5 with sodium hydroxide. Calcium salts may be added
if the level of the free calcium ions is below 50 ppm. The liquefying enzyme
is then added. The slurry is then pumped continuously through a jet cooker
where the temperature is raised to 105o C by direct injection of live stream.
Tremendous shearing forces are exerted on the slurry as it is pumped
through the jet cooker. So in addition to the viscosity reduction action of the
enzyme, some mechanical thinning also occurs. The slurry is maintained at
this high temperature in the pressurized holding cell for about 5 min after
which it is discharged via a spring located release valve into a reaction,
where enzyme action is allowed to continue for about 2 hours at 95o c after
this treatment the liquefied starch will have dextrose equivalent (DE) of
10-20 depending on amount of enzyme used .DE is defined as a reducing
sugars expressed as dextrose and calculated as a percentage of dry
substance. This process is simple energy consumption is relatively low
because the maximum operating temperature is only 105oc as compared to
140-150oc normally used (Borge et al, 1995).
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2.7.1.1

ACID ENZYME LIQUEFACTION:

This

is

another

process

which

takes

advantage

of

the

thermostability B.licheniformis amylase. The amylase is added after the

starch has been cooked and cooled to 100-95oC. Starch slurry containing
30-40% dry solids is cooked at a high temperature for about 5 mins. A jet
cooker is used so that sufficient mechanical thinning due to shearing takes
place (Madsen et al, 1973).
Liquefaction is the first and most important step in starch
processing. The purpose is to provide a partially hydrolysed starch
suspension of relatively low viscosity which is free from by products (Sameh
et al, 2011). Stable to retro gradation and suitable for further processing i.e.,
saccarification with the liquefaction process doesnt go well, problems like
poor filtration and turbidity of the processed solution occurs (Archana et al,
2011). The most important factor for ideal liquefaction of starch is that the
starch slurry which contains suitable amount of alpha amylase is treated at
105- 107oC as quickly and uniformly as possible (Fuentes et al, 2010).
Thermostable amylase is not sufficiently heat stable to be used during
liquefaction process, but they can be used as saccharifying enzymes. The
most widely used enzymes in this group are the maltogenic enzymes (Noda
et al, 2001).
2.7.2

MANUFACTURING OF MALTOSE
Maltose is a naturally occurring disaccharide. Its chemical structure

has

4-0--D-glucopyronosil-D-glucopyranose. It is the main component of

maltose sugar syrup (Yakup et al, 2010). Maltose is widely used as

sweetener and also as intravenous sugar supplement. It is used in food
industry because of low tendency to be crystallized and is relatively non
hygroscopic (Sameh et al, 2011).
Corn, potato, sweet potato and cassava starches are used for maltose
manufacture (Uma et al, 2007). The concentration of starch slurry is
adjusted to be 10-20% for production of medical grade maltose and 20-40%
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for food grade. Thermostable alpha amylase from B.licheniformis and
B.amyloliquefaciens are used (Archana et al, 2011).
2.7.3

MANUFACTURE OF HIGH FRUCTOSE CONTAINING SYRUP

High fructose containing syrups (HFCS) 42 F (Fructose content

equal to 42%) is prepared by enzymic isomerization of glucose with glucose

isomerase. The starch is first converted to glucose by enzyme liquefaction
and saccharification (Shekufeh et al, 2010).
2.7.4

MANUFACTURE OF OLIGOSACCHARIDES MIXTURE

Oligosaccharides mixture (Maltooligomer mixture) is obtained by

digestion of corn starch with alpha amylase, beta amylase and pullulanase.
Maltooligomer mix is a new commercial product. Its composition is usually
as follows: Glucose, 2.2%; maltose, 37.5%; maltotriose, 46.4%; and
maltotetrose and larger malto oligosaccharides, 14% (Marc et al, 2002).
Maltooligomer mix powder obtained by spray drying is highly
hygroscopic. Therefore it serves as a moisture regulator of the food with
which it is mixed (Takata et al, 1992). Maltooligomer mix tastes less sweet
than sucrose. It has lower viscosity than corn syrup because of its low
content of glucose (Shekufeh et al, 2010). Maltooligomer mix is mainly used
as a substitute for sucrose and other saccharides. It is also used for
preventing crystallization of sucrose in foods (Vander et al, 2002).
2.7.5

MANUFACTURE OF MALTOTETROSE SYRUP

Maltotetrose syrup (G4 syrup) is produced by subjecting starch to the

action of maltotetrose forming amylase. The sweetness of the syrup is as low

as 20% of sucrose. Therefore a partial replacement of sucrose with G4 syrup
reduces the sweetness of food without affecting their taste and flavour
(Gulay et al, 2004). It has high moisture retention power which serves to
prevent retrogradation of starch ingredient and retains suitable moisture in
foods (Uma et al, 2007). It has high viscosity than sucrose thus improving
the food texture. G4 syrup can be used to control the freezing points of
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frozen foods. It can be used in industries such as paper sizer (Damien et al,
2010).
2.7.6

MANUFACTURE OF HIGH MOLECULAR WEIGHT BRANCHED

DEXTRINS
Branched dextrins of high molecular weight are prepared by

hydrolysis of corn starch with alpha amylase. The extent of starch

degradation depends on the type of the starch and physical properties
desired (Dhanya et al, 2009). They are obtained as powder after
chromatography and spray drying. These are used as extender and a glozing
agent for production of powdery foods and rice cakes respectively (Harmeet
et al, 2005).
2.7.7

REMOVAL OF STARCH SIZER FROM TEXTILES (DESIZING)

In textiles weaving, starch paste is applied for warping. This gives

strength to textiles at weaving. It also prevents the loss of string by friction,

cutting and generation of static electricity on the string by giving softness to
the surface of the string due to laid down wrap. After weaving the cloth, the
starch is removed and the cloth goes to scouring and dyeing. The starch on
cloth is usually removed by application of alpha amylase (Querong et al,
2008)
2.7.8

DIRECT STARCH FERMENTATION TO ETHANOL

The amylolytic activity rate and amount of starch utilization and

ethanol yields increase in several folds in co cultures (Reeta et al, 2009)

.Moulds amylases are used in alcohol production and brewing industries.
The advantages of such systems are uniform enzyme action in mashes,
increase rate of saccharification, alcohol yield and yeast growth (Maria et al,
2011)

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2.7.9

TREATMENT OF STARCH PROCESSING WASTE WATER (SPW)

Starch is also present in waste produced from food processing plants

(Jamuna

et

al,

1989).Starch

waste

causes

pollution

problems.

Biotechnological treatment of food processing waste water can produce

valuable products such as microbial biomass proteins and also purifies the
effluent (Ashis et al, 2009).

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