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Amylases: an overview with special reference to alpha amylase

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 Journal of Global Biosciences
ISSN 2320-1355
Volume 4, Special Issue 1, 2015, pp. 1886-1901
Website: www.mutagens.co.in
E-mail: [email protected]
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AMYLASES: AN OVERVIEW WITH SPECIAL REFERENCE TO ALPHA

AMYLASE
SP Tiwari1, R Srivastava1, CS Singh1, K Shukla2, RK Singh3, P Singh4, R Singh4, NL Singh5
and R Sharma6
1Department of Microbiology,
VBS Purvanchal University, Jaunpur, UP, India
2Department of Environmental Sciences,

VBS Purvanchal University, Jaunpur, UP, India

3Department of Microbiology,

HNB Garhwal University, Srinagar, UK, India

4 Department of Biological Sciences,

MGCG University, Satna, MP, India

5 Department of Chemistry,

Faculty of Science, Banaras Hindu University, Varanasi, India

6Department of Biotechnology,

VBS Purvanchal University, Jaunpur, UP, India.

INTRODUCTION
Enzymes are biological catalysts which regulate specific biochemical reactions. In recent years,
the potential of using microorganisms as biotechnological sources of industrially relevant
enzymes has stimulated interest in the exploration of extracellular enzymatic activity in several
microorganisms. Among the industrially important enzymes, proteases and amylases are
considered to be the most prominent enzymes since they are widely utilized in brewing,
detergent, and food industries[1]. Amylases are employed in the starch processing industries for
the hydrolysis of polysaccharides such as starch into simple sugar constituents. With the advent
of new frontiers in biotechnology, the spectrum of amylase applications has expanded into
many new fields such as clinical, medicinal, and analytical chemistry[2].
Starch processing, which is undertaken in two steps, involves liquefaction of the polysaccharide
using bacterial α-amylase, followed by saccharification catalyzed by fungal glucoamylase. After
the World War II, enzyme applications increased due to advances in industrial microbiology and
biochemical engineering[3]. Now a days, enzymes are employed in many different areas such as
food, feed, detergent, textiles, laundry, tanning, as well as pharmaceuticals, cosmetics, and fine-
chemicals industries. Industrial applications account for over 80% of the global market of
enzymes[4]. At least 50% of the enzymes marketed today are obtained from genetically modified
organisms, employing genetic and protein engineering. Food enzymes are the most widely used
and still represent the major share in enzyme market.
The history of amylases began in 1811 when the first starch degrading enzyme was discovered
by Kirchhoff in wheat and laid down the foundation for the discovery and research on Amylase.
The α-amylases were named by Kuhn in 1925, because the hydrolysis products are in the alpha
configuration. In 1930, Ohlsson discovered another amylase, which yielded a β-mannose. He
named it β-amylase. Crystal structure was established using 3Å resolution structure which was
further improved to a 1.5 Å resolution of α-amylases[5]. The three dimensional crystal structures
of each form were determined in the 1990s and found to be effectively identical[6].
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As diastase, amylase was the first enzyme to be discovered and isolated by Anselme Payen,
1833[7]. Interestingly, the first enzyme produced industrially was an amylase from a fungal
source in 1894, which was used as a pharmaceutical aid for the treatment of digestive
disorders[8]. Boidin & Effront, 1917 were the first to use Bacillus. subtilis and Bacillus
mesentericus for the production of α-amylases on commercial scale using large fermentors in
submerged fermentation[9]. Employment of bacterial cultures for the production of commercial
enzyme was pioneered by them and accepted as an industrial practice throughout the world for
the production of bacterial α-amylases. Prior to the developments, fungal amylases were
extensively produced in the United States by the SSF techniques as pioneered by Takamine[10].
Types of Amylases
Enzymes belonging to amylases, endoamylases and exoamylases, are able to hydrolyse starch.
These enzymes are classified according to the manner in which the glycosidic bond is attacked.
The starch degrading enzymes are found in the numerous glycoside hydrolase families 13(GH-
13 families)[11-12].
α- Amylase (EC 3.2.1.1)
Endoamylases are able to cleave α,1-4 glycosidic bonds present in the inner part (endo-)
of the amylose or amylopectin chain. α-amylase is a well-known endoamylase. It is found in a
wide variety of microorganisms, belonging to the Archaea as well as the bacteria[13]. The end
products of α-amylase action are oligosaccharides with varying length with α-configuration and
α-limit dextrins, which constitute branched oligosaccharides. α- amylases are often divided into
two categories according to the degree of hydrolysis of the substrate[14]. Saccharifying α-
amylases hydrolyze 50 to 60% and liquefying α-amylases cleave about 30 to 40% of the
glycosidic linkages of starch. The α-amylases are calcium metalloenzymes, completely unable to
function in the absence of calcium., α-amylase breaks down long-chain carbohydrates by acting
at random locations along the starch chain, ultimately yielding maltotriose and maltose from
amylose, or maltose, glucose and "limit dextrin" from amylopectin. α-amylase tends to be faster-
acting than ß-amylase because it can act anywhere on the substrate. In human physiology, both
the salivary and pancreatic amylases are α-Amylases and are also found in plants (adequately),
fungi (ascomycetes and basidiomycetes) and bacteria (Bacillus).
β- Amylase (E.C. 3.2.1.2)
Enzymes belonging to the second group, the exoamylases, either exclusively cleave α,1-4
glycosidic bonds such as ß-amylase or cleave both α,1-4 and α,1-6 glycosidic bonds like
amyloglucosidase or glucoamylase (E.C. 3.2.1.3) and α-glucosidase (E.C. 3.2.1.20). Exoamylases
act on the external glucose residues of amylose or amylopectin and thus produce only glucose
(glucoamylase and α-glucosidase), or maltose and ß-limit dextrin. ß-amylase and glucoamylase
also convert the anomeric configuration of the liberated maltose from α to ß. Glucoamylase and
α-glucosidase differ in their substrate preference, α-glucosidase acts best on short
maltooligosaccharides and liberates glucose with α-configuration while glucoamylase
hydrolyzes long-chain polysaccharides best. β-amylases and glucoamylases have also been
found in a large variety of microorganisms[13].
γ-Amylase (EC 3.2.1.3)
γ-amylase cleaves α(1-6) glycosidic linkages, in addition to cleaving the last α (1-4)
glycosidic linkages at the non-reducing end of amylose and amylopectin, yielding glucose.
Unlike the other forms of amylase, γ-amylase is most efficient in acidic environments and has an
optimum pH of 3.
Sources of Alfa Amylase
Plant sources
Amylases are widespread in animals, fungi, plants, and are also found in the unicellular
eukaryotes, bacteria and archaea[15]. Though plants and animals produce amylases, enzymes
from microbial sources are generally used in industrial processes. This is due to a number of
factors including productivity, thermostability of the enzyme as well as ease of cultivating
microorganisms[16]. The major advantages of the enzymatic route are the selectivity with its
associated high yield and exclusivity toward the desired product[17]. Bacteria used in
commercial production are the Bacillus spp. [18-19, 13]. Others, such as Escherichia spp,

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Pseudomonas, Proteus, Serratia and Rhizobium also yield appreciable quantity of the enzyme [20].
Aspergillus, Rhizopus, Mucor, Neurospora, Penicillium and Candida are some of the fungi that also
produce extracellular amylases of commercial value [19]. Plant sources had not been considered
with enough significance as the source of these enzymes yet [21]. The utilization of agriculture
waste materials serves two functions: reduction in pollution and upgrading of these materials.
Agricultural wastes are being used for both liquid and solid fermentation to reduce the cost of
fermentation media. These wastes consist of carbon and nitrogen sources necessary for the
growth and metabolism of organisms. These nutrient sources included pearl millet starch,
orange waste, potato, corn, tapioca, wheat and rice as flours were used for α-amylase
production [22-23]. α-Amylases are being produced commercially in bulk from microorganisms
and represent about 25-33% of the world enzyme market[24]. They had numerous applications
including liquefaction of starch in the traditional beverages, baking and textile industry for
desizing of fabrics [25-26]. Moreover, they have been applied in paper manufacture, medical fields
as digestives and as detergent additives [27-28].
Animal sources
Ptyalin, a salivary α-amylase (α-1,4-α-D-glucan-4-glucanohydrolase; E.C. 3.2.1.1) is one
of the most important enzymes in saliva. The enzyme was first described in saliva by Leuchs in
1831[29]. It consists of two families of isoenzymes, of which one set is glycosylated and the other
contains no carbohydrate. The molecular weight of the glycosylated form is about 57 kDa; that
of the non-glycosylated form is about 54 kDa. Salivary amylases accounts for 40% to 50% of the
total salivary protein and most of the enzyme being synthesized in the parotid gland (80% of
the total)[29-30]. It is a calcium-containing metalloenzyme that hydrolyzes the α-1,4 linkages of
starch to glucose and maltose. It is known to be mainly involved in the initiation of the digestion
of starch in the oral cavity. However, Salivary α-amylase has also been shown to have an
important bacterial interactive function[31].
Microbial sources
In spite of the wide distribution of amylases, microbial sources, mainly fungal and
bacterial amylases, are used for the industrial production due to advantages such as cost
effectiveness, consistency, less time and ease of process modification and optimization [32-33].
Fungal amylases have been widely used for the preparation of oriental foods [34]. Among
bacteria, Bacillus sp. is widely used for thermostable α-amylase production to meet industrial
needs. B. subtilis, B. stearothermophilus, B. licheniformis and B. amyloliquefaciens are known to
be good producers of α-amylase and these have been widely used for commercial production of
the enzyme for various applications [35]. Similarly, filamentous fungi have been widely used for
the production of amylases for centuries. As these moulds are known to be prolific producers of
extracellular proteins, they are widely exploited for the production of different enzymes
including α-amylase.
Fungal amylases
Fungi belonging to the genus Aspergillus have been most commonly employed for the
production of α-amylase. With the development of genetic engineering, Bacillus subtilis is
becoming an increasingly attractive host for cloning. The advantages of B. subtilis such as high
secretion level and non-pathogenic safe (GRAS-generally recognized as safe) status for non-
antibiotics strains have made it suitable for the production of heterologous enzymes [36-37]. Most
reports about fungi that produce α-amylase have been limited to a few species of mesophilic
fungi, and attempts have been made to specify the cultural conditions and to select superior
strains of the fungus to produce on a commercial scale [19]. Fungal sources are confined to
terrestrial isolates, mostly to Aspergillus and Penicillium [38]. The Aspergillus species produce a
large variety of extracellular enzymes, and amylases are the ones with most significant
industrial importance [39]. Filamentous fungi, such as Aspergillus oryzae and Aspergillus niger,
produce considerable quantities of enzymes that are used extensively in the industry. A. oryzae
has received increased attention as a favorable host for the production of heterologous proteins
because of its ability to secrete a vast amount of high value proteins and industrial enzymes, e.g.
α-amylase [40]. Aspergillus oryzae has been largely used in the production of food such as soy
sauce, organic acid such as citric and acetic acids and commercial enzymes including α-amylase

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[41]. Aspergillus niger has important hydrolytic capacities in the α-amylase production and, due
to its tolerance of acidity (pH < 3), it allows the avoidance of bacterial contamination [42].
Filamentous fungi are suitable microorganisms for solid state fermentation (SSF), especially
because their morphology allows them to colonize and penetrate the solid substrate [43]. The
fungal α-amylases are preferred over other microbial sources due to their more accepted GRAS
status [19]. The thermophilic fungus Thermomyces lanuginosus is an excellent producer of
thrmostable amylase purified the α-amylase, proving its thermostability [44-45].
Bacterial amylases
The production of microbial amylases from bacteria is dependent on the type of strain,
composition of medium, method of cultivation, cell growth, nutrient requirements, incubation
period, pH, temperature, metal ions and thermostability. In fact, such industrially important
microorganisms found within the genus Bacillus, can be exploited commercially due to their
rapid growth rate leading to short fermentation cycles, capacity to secrete proteins into the
extracellular medium and safe handling [13].
α-Amylase can be produced by different species of microorganisms, but for commercial
applications α-amylase is mainly derived from the genus Bacillus. α-Amylases produced from
Bacillus licheniformis, Bacillus stearothermophilus, and Bacillus amyloliquefaciens find potential
application in a number of industrial processes such as in food, fermentation, textiles and paper
industries[46, 13]. Thermostability is a desired characteristic of most of the industrial enzymes.
Thermostable enzymes isolated from thermophilic organisms have found a number of
commercial applications because of their stability. As enzymatic liquefaction and
saccharification of starch are performed at high temperatures (100–110°C), thermostable
amylolytic enzymes have been currently investigated to improve industrial processes of starch
degradation and are of great interest for the production of valuable products like glucose,
crystalline dextrose, dextrose syrup, maltose and maltodextrins [47-49]. Bacillus subtilis, Bacillus
stearothermophilus, Bacillus licheniformis, and Bacillus amyloliquefaciens are known to be good
producers of thermostable α-amylase, and these have been widely used for commercial
production of the enzyme for various applications [50]. Thermostable α-amylases have been
reported from several bacterial strains and have been produced using SmF as well as SSF
[51].However, the use of SSF has been found to be more advantageous than SmF and allows a

cheaper production of enzymes [52]. The production of α-amylase by SSF is limited to the genus
Bacillus, and B. subtilis, B. polymyxia, B. mesentericus, B. vulgarus, B. megaterium and B.
licheniformis have been used for α-amylase production in SSF [53]. Currently, thermostable
amylases of Bacillus stearothermophilus or Bacillus licheniformis are being used in starch
processing industries [48]. Enzymes produced by some halophilic microorganisms have optimal
activity at high salinities and could therefore be used in many harsh industrial processes where
the concentrated salt solutions used would otherwise inhibit many enzymatic conversions [54,
50]. In addition, most halobacterial enzymes are considerably thermotolerant and remain stable

at room temperature over long periods. Halophilic amylases have been characterized from
halophilic bacteria such as Chromohalobacter sp. [50], Halobacillus sp. [54], Haloarcula hispanica
[55], Halomonas meridian [56] and Bacillus dipsosauri [57].

Bacillus is endowed to produce thermostable α-amylase and also large quantities of

other enzymes. Indeed, 60% of commercially available enzymes are obtained from different
species of Bacillus i.e. B. subtilis, B. stearothermophilus, B. licheniformis and B. amyloliquefaciens
[33]. Some Bacillus strains produce enzyme in the exponential phase, whereas some others in the

mid stationary phase. Though, different Bacillus species have similar growth patterns and
enzyme profiles, but their optimized conditions vary, depending upon the strain. Some
properties exhibited by different bactria are shown in following table-

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Table: Bacterial α-amylases & their characteristics

Mode pH Temp Mol. Inhibitors Ref.
Microorganisms of optim . Wt.
Ferme a optim (kDa)
n- a
tation
Chromohalobacter sp. SSF 7.0 - 9.0 65 °C 72 - [50
TVSP 101 ]
Haloarcula hispânica 6.5 50 °C 43.3 EDTA [50
]
Bacillus sp. I-3 SmF 7.0 70 °C - EDTA, HgCl2 [58
]
Bacillus sp. PN5 SmF 10 60 °C - NH4Cl [59
]
Bacillus subtilis DM-03 SSF 6.0–10 50 °C - - [60
]
Bacillus subtilis SmF 6.5 37 °C - - [61
KCC103 ]
Bacillus sp. KCA102 7.1 57.5 - - [62
°C ]
Bacillus subtilis JS- SmF 7.0 50 °C Co2+ Cu2+ Hg2+ Mg2+ [47
2004 - ]
Zn2+ Ni2+ Fe2+ and
Mn2+
Bacillus subtilis SmF 7.0 135 - - [46
°C ]
Bacillus caldolyticus SmF 5.0-6.0 70 °C - - [63
DSM405 ]
Bacillus sp. 4.5 70 °C 53 Hg2+Zn2+ EDTA [64
Ferdowsicous ]
Halomonas meridian SmF 7.0 37 °C - Glu [56
]
Geobacillus - 7.0 70 °C - - [65
thermoleovorans ]

Mode of Action of Alfa Amylase

In general, it is believed that α-amylases are endo-acting amylases which hydrolyze α- (1-4)
glycosidic bonds of the starch polymers internally. Several models for amylase action pattern
have been proposed, such as the random action and the multiple attack action. Random action
has also been referred to as a single attack or multi-chain attack action [13]. In the former, the
polymer molecule is successively hydrolysed completely before dissociation of the enzyme-
substrate complex. While, in the latter, only one bond is hydrolysed per effective encounter. The
multiple attack action is an intermediate between the single-chain and the multi-chain action
where the enzyme cleaves several glycosidic bonds successively after the first (random)
hydrolytic attack before dissociating from the substrate. It is observed that the multiple attack
action is generally an accepted concept to explain the differences in action pattern of amylases
[66]. However, most of the endoamylases have a low to very low level of multiple attack action

increased with temperature to a degree depending on the amylase itself.

Structure of Alfa Amylase
Molecular weight
Despite wide difference of microbial α-amylases characters, their molecular weights are
usually in the same range 40-70 kDa [19] while the highest molecular weight of α-amylases, 210
kDa, for Chloroflexus aurantiacus [67]. Whereas, 10 kDa of Bacillus caldolyticus α-amylase was

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reported to be the lowest value [19]. This molecular weight may be raised due to glycosylation as
in the case of Thermoactiomyces vulgaris α-amylase that reach 140 kDa [68]. In contrast,
proteolysis may lead to decrease in the molecular weight. For example, α-amylase of T. vulganis
94-2A (AmyTV1) is a protein of 53 kDa and smaller peptides of 33 and 18 kDa that have been
shown to be products of limited AmyTV1 proteolysis [69].
Molecular structure
α-amylases from different organisms share about 30% amino acid sequence identity and
all belong to the same Glycosyl Hydrolase family 13 (GH-13 family of protein) [70]. The three
dimensional (3D) structures of α-amylases have revealed monomeric, calcium-containing
enzymes, with a single polypeptide chain folded into three domains (A-C). The most conserved
domain in α-amylase family enzymes, the A-domain, consists of a highly symmetrical fold of
eight parallel ß-strands arranged in a barrel encircled by eight α-helices. The highly conserved
amino acid residues of the α-amylase family involved in catalysis and substrate binding are
located in loops at the C-termini of ß-strands in this domain. This is typical to all enzymes
belonging to the α/ß –barrel protein family [71]. α-amylases have a B-domain that protrudes
between ß-sheet number 3 and α-helix number. 3. It ranges from 44 to 133 amino acid residues
and plays a role in substrate or Ca binding. The sequence of this domain varies most; in Bacillus,
α-amylases it is relatively long and folds into a more complex structure of ß-strands [72],
whereas in barley α-amylase there is an irregularly structured domain of 64 residues. All known
α-amylases, with a few exceptions, contain a conserved Ca+2 binding site which is located at the
interface between domains A and B [73]. In addition, α-amylase produced by Bacillus
thermooleovorans was found to contain a chloride ion binding site in their active site [74], which
has been shown to enhance the catalytic efficiency of the enzyme, presumably by elevating the
pKa of the hydrogen-donating residue in the active site [50].
α-amylases has a domain C which is relatively conserved and folds into an antiparallel ß
barrel. The orientation of domain C relative to domain A varies depending on the type and
source of amylase [75]. The function of this domain is unknown. Structural studies have
confirmed that the active sites of glycosyl hydrolases are composed of multiple binding sites, or
subsites, for the sugar units of polymeric substrates. The open active site cleft is formed
between domains A and B, so that residues from domain B participate in substrate binding. The
substrate binding sites are commonly lined with aromatic residues (Phe, Trp and Tyr) which
make hydrophobic stacking interactions with the sugar rings. In addition, the active sites
contain many residues which form hydrogen bonds to the substrate either directly or via water
molecules [76, 66 ]. In Taka-amylase A, the first examined protein α-amylase by X-ray
crystallography, three acidic residues, i.e., one glutamic and two aspartic acids were found at the
centre of the active site, and subsequent mutational studies have shown that these residues are
essential for catalysis [77, 66]. The glutamic acid residue is now believed to be the proton donor,
while the first of the two conserved aspartic acids appearing in the amino acid sequence of an α-
amylase family member is thought to act as the nucleophile. The role of the second aspartic acid
is less certain, but it has been suggested to involved in stabilising the oxocarbenium ion-like
transition state and also in maintaining the glutamic acid in the correct state of protonation for
activity [78]. These residues occur near the ends of strands 3, 4, 5 and 7 of the a/ß -barrel and are
found in four short sequences, long-recognised as being conserved in α-amylase family
enzymes.
Applications of Alfa 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

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number of common characteristics such as α (ß/α)8 barrel structure, the hydrolysis or

formation of glycosidic bonds in the a 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 [79]. 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 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 flavor [80].
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 [81, 82].
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 [83-85]. Many
industries used alpha amylases for the production of glucose. Enzyme hydrolyzed the starch and
converted it into glucose. They hydrolyze α-1,4 glucosidic linkage in the starch polymer in a
random manner to yield glucose and maltose [86]. Therefore, alpha amylase is extensively used
in many industries for the production of glucose and also used in water-soluble dextrin [87].
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 [88].
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[89]. Examples of amylases obtained from microorganisms used in

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paper industry includes Amizyme® (PMP Fermentation Products, Peoria,USA), Termamyl®,

Fungamyl, BAN® (Novozymes,Denmark) and α-amylase G9995® (Enzyme Biosystems, USA)
[90].

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 [91, 92]. The addition of enzyme stabilizes the bleach agent and preserves effectiveness
of the bleach in laundry detergent bar composition [93, 94]. 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 emperature 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 -185°F 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 [95].
Fuel alcohol production
Ethanol is the most utilized liquid biofuel. For the ethanol production, starch is the most
used substrate due to its low price and easily available raw material in most regions of the
world [96]. In this production, starch has to be solubilized and then submitted to two enzymatic
steps in order to obtain fermentable sugars. The bioconversion of starch into ethanol involves
liquefaction and saccharification, where starch is converted into sugar using an amylolytic
microorganism or enzymes such as α-amylase, followed by fermentation, where sugar is
converted into ethanol using an ethanol fermenting microorganism such as yeast
Saccharomyces cerevisiae [97, 98]. The production of ethanol by yeast fermentation plays an
important role in the economy of Brazil [99]. In order to obtain a new yeast strain that can
directly produce ethanol from starch without the need for a separate saccharifying process,
protoplast fusion was performed between the amylolytic yeast Saccharomyces fibuligera and S.
cerevisiae [96]. Among bacteria,α-amylase obtained from thermoresistant bacteria like Bacillus
licheniformis or from engineered strains of Escherichia coli or Bacillus subtilis is used during the
first step of hydrolysis of starch suspensions [100].
Treatment of starch processing waste water (SPW)
Starch is also present in waste produced from food processing plants. Starch waste
causes pollution problems. Biotechnological treatment of food processing waste water can
produce valuable products such as microbial biomass protein and also purifies the effluent [101-
104].

Analysis in medicinal and clinical chemistry

With the advent of new frontiers in biotechnology, the spectrum of amylase applications
has expanded into many other fields, such as clinical, medicinal and analytical chemistry. There
are several processes in the medicinal and clinical areas that involve the application of
amylases. The application of a liquid stable reagent, based on α-amylase for the Ciba Corning
Express clinical chemistry system has been described [105]. A process for the detection of higher
oligosaccharides, which involved the application of amylase was also developed [106]. This
method was claimed to be more efficient than the silver nitrate test. Biosensors with an
electrolyte isolator semiconductor capacitor (EIS-CAP) transducer for process monitoring were
also developed [107].
Other applications

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• An inhibitor of alpha-amylase called phaseolamin has been tested as a potential diet aid
[108].

• A higher than normal concentration of amylases may reflect one of several medical
conditions, including acute inflammation of the pancreas (concurrently with the more
specific lipase), but also perforated peptic ulcer, torsion of an ovarian cyst, strangulation
ileus, macroamylasemia and mumps. Amylase may be measured in other body
fluids,including urine and peritoneal fluid.
• In molecular biology, the presence of amylase can serve as an additional method of
selecting for successful integration of a reporter construct in addition to antibiotic
resistance. As reporter genes are flanked by homologous regions of the structural gene
for amylase, successful integration will disrupt the amylase gene and prevent starch
degradation, which is easily detectible through iodine staining.

REFERENCES
1. Doss, A & Anand, SP. 2012. Purification and characterization of extracellular amylolytic
enzyme from Aspergillus species. Afr. J. Biotechnol. 11(83): 14941-14945.

2. Khan, JA & Yadav, SK. 2011. Production of alpha amylases by Aspergillus niger using cheaper
substrates employing solid state fermentation. Int. J. Pl.An. Env. Sci. 1(3): 100-108.

3. Illanes, A. Introduction. In; Illanes A. (ed.).2008. Enzyme Biocatalysis. Principles and

Applications. Springer. :19-56.

4. Van Oort, M. 2010. Enzymes in food technology – introduction. In: Whitehurst RJ, van Oort
M. (eds.) Enzymes in Food Technology, second ed. Chichester: Wiley-Blackwell. : 1-17.

5. Kumari, A; Singh, S & Kayastha, AM. 2012. α-Amylase: General properties,machenism and
Biotechnological application-A Review. Current Biotechnology. 1: 98-197.

6. Qian, M; Haser, R & Payan, F. 1995.Corbohydrate Binding sites in a pancreatic α-amylase

substrate complex,derived from X-ray structute analysis at 2.1 A resolution. Protein
Sci.4,747.

7. Hill, R & Needham, J Ed.1970. The Chemistry of Life: Eight Lectures on the History of
Biochemistry, Cambridge University Press, London, England, 17.

8. Pandey, A; Nigam, P; Soccol, CR; Soccol, VT; Singh, D & Mohan, R. 2000. Advances in
microbial amylases. Biotechnol Appl Biochem. 31(2):135-152.

9. Boidin, A & Effront, J. 1917, US Patent 1, 227, 374, May 22, 1917.

10. Takamine, J.1914. Enzymes of Aspergillus oryzae and the application of its amyloclastic
enzyme to the fermentation industry. Ind. Eng. Chem. 6:824-828.

11. Coutinho, P & Henrissat, B. 1999. Carbohydrate active enzymes. In: Recent Advances in
Carbohydrate bioengineering, H. Gilbert, G. Davies, B. Svensson & B. Henrissat (Ed.). Royal
Society of Chemistry, Cambridge.:3-12.

12. Henrissat, B & Bairoch, A. 1993. New families in the classification of glycosyl hydrolases
based on amino acid sequence similarities. Biochem. J. 293(3):781-788.

13. Pandey, A; Nigam, P; Soccol, CR; Soccol, VT; Singh, D & Mohan, R. 2000. Advances in
microbial amylases. Biotechnol Appl Biochem. 31(2):135-152.

http://mutagens.co.in 1894
Journal of Global Biosciences Vol. 4, SI 1, 2015 pp. 1886-1901
ISSN 2320-1355

14. Fukumoto, J & Okada, S. 1963. Studies on bacterial amylase, Amylase types of Bacillus
subtilis species. J. Ferment. Technol. 41:427-434.

15. Da Lagea, JL ;Etienne, GJ; Danchinc, EGJ & Casane, D. 2007. Where do animal α-amylases
come from? An interkingdom trip. Feder. Eur.Biochem. Soc. 581:3927–3935.

16. Reddy, R; Reddy, G & Seenayya, G. 1999. Enhanced production of thermostable α-amylase of
pullulunase in the presence of surfactants by Clostridium thermosulfurogenes SV2. Process
Biochem. 34: 87-92.

17. Kim, S & Dale, BE. 2004. Global potential bioethanol production from wasted crops and
crops residues. Biomass Bioeng. 26:361-375.

18. Olafimihan, C & Akinyanju, J. 1999. Thermophilic amylase producers from the soil. Nigeria. J.
Pure Appl. Sci. 14: 816-822.

19. Gupta, R; Gigras, P; Mohapatra, H; Goswami, VK & Chauhan, B. 2003. Microbial α-amylases: a
biotechnological perspective. Process Biochem. 38:1599 - 1616.

20. Oliviera, A; Oliviera, L; Andrade, J & Chagas, A. 2007. Rhizobia amylase production using
various starchy substances as carbon source. Braz.J. Microbiol. 38:1599-1616.

21. Azad, MAK; Bae, JH; Kim, JS; Lim, JK; Song, KS; Shin, BS & Kim, HR. 2009. Isolation and
characterization of a novel thermostable amylase from Korean pine seeds. New Biotechnol.
26:143-149.

22. Djekrif-Dakhmouche, S; Gheribi-Aoulmi, Z; Meraihi, Z & Bennamoun, L. 2006. Application of

a statistical design to the optimization of culture medium for α-amylase production by
Aspergillus niger ATCC 16404 grown on orange waste powder. J. Food Eng. 73: 190-197.

23. Haq, I; Ashraf, H; Qadeer, MA & Iqbal, J. 2005. Pearl millet, a source of alpha amylase
production by Bacillus licheniformis. Bioresource.Technol. :1201-1204.

24. Nguyen, QD; Rezessy-Szabo, JM; Claeyssens, M; Stals, I & Hoschke, A. 2002. Purification and
characterization of amylolytic enzymes from thermophilic fungus Thermomyces lanuginosus
strain ATCC 34626. Enzyme Microb. Technol. 31: 345-352.

25. Dauter, Z; Dauter, M; Brzozowski, AM; Christensen, S; Borchert, TV; Beier, L; Wilson, KS &
Davies, G. 1999. X-ray structure of novamyl, the fivedomain “maltogenic”α-amylase from
Bacillus stearothermophilus:maltose and acarbose complexes at 1.7 A resolution. Biochem.
38:8385-8392.

26. Pederson, H & Neilsen, J. 2000. The influence of nitrogen sources on α-amylase productivity
by Aspergillus oryzae in continous cultures. Applied Microbiol and Biotechnol. 53:278-281.

27. Bruinenberg, P; Hulst, A; Faber, A & Voogd, R. 1996. A process for surface sizing or coating of
paper. Eur. Patent Appl. EP 0,690,170 A1.

28. Mitidieri, S; Martinelli, AHS; Schrank, A & Vainstein, MH. 2006. Enzymaticdetergent
formulation containing amylase from Aspergillus niger: a comparative study with
commercial detergent formulations. Biores. Technol. 97:1217-1224.

http://mutagens.co.in 1895
Journal of Global Biosciences Vol. 4, SI 1, 2015 pp. 1886-1901
ISSN 2320-1355

29. Zakowski, JJ & Bruns, DE. 1985. Biochemistry of human alpha amylase isoenzymes. Crit.
Rev. Clin. Lab. Sci. 21:283-322.

30. Makinen, KK. 1989. Salivary enzymes. In: Tenovuo, J.O. (Ed.),Human Saliva: Clinical
Chemistry and Microbiology, vol. 2. CRC Press, Boca Raton. :93-119.

31. Cannapieco, FA; Torres, G & Levine, MJ. 1993. Salivary alpha amylase: role in dental plaque
and caries formation. Crit. Rev.Oral Biol. Med. 4:301-307.

32. Mamo, G & Gessesse, A. 1999. Effect of cultivation conditions on growth and alpha
Production. Biotechnol Tech. 11:447-450.–

33. Burhan, A; Nisa, U; Gokhan, C; Omer, C; Ashabil, A & Osman, G. 2003. Enzymatic properties
of a novel thermostable,thermophilic, alkaline and chelator resistant amylase from an
alkaliphilic Bacillus sp. Isolate ANT-6. Process. Biochem. 38:1397–1403.

34. Ivanova, V; Yankov, D; Kabaivanova, L & Pashkoulov, D. 2001. Simultaneous biosynthesis

and purification of two extracellular Bacillus hydrolases in aqueous two alpha–amylase.
Biochem. Eng. J. 8:61–81.

35. Kim, TU; Gum, GB; Jeong, JY & Young, SC. 1995. Purification and characterization of a
maltotetraose forming alkaline alpha amylase from an alkalophilic Bacillus strain, GM 8901.
Appl. Environ. Microbiol. 61:3105–3112.

36. Sharp, RJ; Scawen, MD & Atkinson, T. 1989.Fermentation and downstream processing of
Bacillus. In: Harwood CR (ed) Bacillus.Biotechnology handbooks. Plenum Press, NewYork.
10:255–288.

37. Deboer, AS & Diderichsen, B.1991. On the safety of Bacillus subtilis and B.amyloliquefaciens.
a review. Appl. Microbiol.Biotechnol. 36:1–4.

38. Kathiresan, K & Manivannan, S. 2006. α-Amylase production by Penicillium fellutanum

isolated from mangrove rhizosphere soil. Afr. J.Biotechnol. 5:829-832.

39. Hernandez, MS; Rodriguez, MR; Perez-Guerra, N & Perez-Roses, R. 2006. Amylase
production by Aspergillus niger in submerged cultivation on two wastes from food
industries. Journal of food engg. 73:93-100.

40. Jin, B; van Leeuwen, HJ; Patel, B & Yu, Q.1998. Utilisation of starch processing wastewater
for production of microbial biomass protein and fungal α-amylase by Aspergillus oryzae.
Bioresour. Technol. 66:201-206.

41. Kammoun, R; Naili, B & Bejar, S. 2008. Application of a statisticaldesign to the optimization
of parameters and culture medium for alpha amylase production by Aspergillus oryzae CBS
819.72 grown on gruel(wheat grinding by-product). Bioresour Technol. 99:5602-5609.

42. Enshasy, H E1; Abuoul-Enein, A; Helmy, S & Azaly Y El. 2008. Optimization of The Industrial
Production of Alkaline Protease By Bacillus licheniformis In Different Production Scales.
Australian Journal of Basic and Applied Sciences. 2:583-593.

43. Rahardjo, YSP; Weber, FJ; Haemers, S; Tramper, J & Rinzema, A. 2005. Aerial mycelia of
Aspergillus oryzae accelerate α-amylase production in a model solid-state fermentation
system. Enzyme Microb.Technol. 36: 900–902.

http://mutagens.co.in 1896
Journal of Global Biosciences Vol. 4, SI 1, 2015 pp. 1886-1901
ISSN 2320-1355

44. Jensen, B; Nebelong, P; Olsen, J; Reeslev, M. 2002. Enzyme production in continuous

cultivation by the thermophilic fungus,Thermomyces lanuginosus. Biotechnology Letters.
24: 41–45.

45. Kunamneni, A; Permaul, K & Singh, S. 2005. Amylase production in solid state fermentation
by the thermophilic fungus Thermomyces lanuginosus. J Biosci Bioeng. 2: 168-171.

46. Konsoula, Z & Liakopoulou-Kyriakides, M. 2007. Co-production of alpha-amylase and beta-

galactosidase by Bacillus subtilis in complex organic substrates. Bioresour Technol. 98
:150-157.

47. Asgher, M; Asad, MJ; Rahman, SU & Legge, RL. 2007. A thermostable α-amylase from a
moderately thermophilic Bacillus subtilis strain from starch processing. J.Food Eng. 79:950-
955.

48. Gomes, I; Gomes, J & Steiner, W. 2003. Highly thermostable amylase and pullulanase of the
extreme thermophilic eubacterium Rhodothermus marinus. production and partial
characterization. Bioresour Technol. 90:207-214.

49. Stamford, TL; Stamford, NP; Coelho, LC & Araujo, M. 2001. Production and characterization
of a thermostable alpha-amylase from Nocardiopsis sp. endophyte of yam bean. Bioresour
Technol. 76: 137-141.

50. Prakash, O & Jaiswal, N. 2009. Alpha-Amylase: An Ideal Representative of Thermostable

Enzymes. Appl Biochem Biotechnol. 160(8):2401-2414.

51. Teodoro, CES & Martins, MLL. 2000. Culture conditions for the production of thermostable
amylase by Bacillus sp. Braz. J. Microbiol. 31:298-302.

52. Sodhi, HK; Sharma, K; Gupta, JK & Soni, SK. 2005. Production of a thermostable α-amylase
from Bacillus sp. PS-7 by solid state fermentation and its synergistic use in the hydrolysis of
malt starch for alcohol production. Process Biochem. 40: 525–534.

53. Baysal, Z; Uyar, F & Aytekin, C. 2003. Solid state fermentation for production of α-amylase
by a thermotolerant Bacillus subtilis from hotspring water. Process Biochemistry. 38:1665-
1668.

54. Amoozegar, MA; Malekzadeh, F & Malik, KA.2003. Production of amylase by newly isolated
moderate halophile, Halobacillus sp. Strain MA-2. J Microbiol Methods. 52:353-359.

55. Hutcheon, GW; Vasisht, N & Bolhuis, A. 2005. Characterisation of a highly stable alpha-
amylase from the halophilic archaeon Haloarcula hispanica. Extremophiles. 9: 487-495.

56. Coronado, M; Vargas, C; Hofemeister, J; Ventosa, A & Nieto, JJ. 2000. Production and
biochemical characterization of an alpha-amylase from the moderate halophile Halomonas
meridiana. FEMS Microbiol Lett. 183:67-71.

57. Deutch, CE. 2002. Characterization of a salt-tolerant extracellular α-amylase from Bacillus
dipsosauri. Lett Appl Microbiol. 35:78-84.

58. Goyal, N; Gupta, JK & Soni, SK. 2005. A novel raw starch digesting thermostable α-amylase
from Bacillus sp. I-3 and its use in the direct hydrolysis of raw potato starch. Enzyme
Microb. Technol. 37:723–734.

http://mutagens.co.in 1897
Journal of Global Biosciences Vol. 4, SI 1, 2015 pp. 1886-1901
ISSN 2320-1355

59. Saxena, RK; Dutt, K; Agarwal, L & Nayyar, P. 2007. A highly thermostable and alkaline
amylase from a Bacillus sp. PN5. Bioresour Technol. 98:260-265.

60. Mukherjee, AK; Borah, M & Raí, SK. 2009. To study the influence of different components of
fermentable substrates on induction of extracellular α-amylase synthesis by Bacillus subtilis
DM-03 in solidstate fermentation and exploration of feasibility for inclusion of amylase in
laundry detergent formulations. Biochem. Eng. J. 43:149-156.

61. Rajagopalan, G & Krishnan, C. 2008. Alpha-amylase production from catabolite derepressed
Bacillus subtilis KCC103 utilizing sugarcane bagasse hydrolysate. Bioresour Technol.
99:3044-3050.

62. Agrawal, M; Pradeep, S; Chandraraj, K & Gummadi, SN. 2005. Hydrolysis of starch by
amylase from Bacillus sp. KCA102.a statistical approach. Process Biochemistry. 40:2499-
2507.

63. Schwab, K; Bader, J; Brokamp, C; Popovic, MK; Bajpa, R & Berovic, M. 2009. Dual feeding
strategy for the production of alpha-amylase by Bacillus caldolyticus using complex media.
N Biotechnol. 26:68-74.

64. Asoodeh, A; Chamani, J & Lagzian, M. 2010. A novel thermostable, acidophilic alpha-amylase
from a new thermophilic "Bacillus sp.Ferdowsicous" isolated from Ferdows hot mineral
spring in Iran:Purification and biochemical characterization. Int J Biol Macromol., 46.
:289–297.

65. Rao, JLUM & Satyanarayana, T. 2007. Improving production of hyperthermostable and high
maltose-forming α-amylase by an extreme thermophile Geobacillus thermoleovorans using
response surface methodology and its applications. Bioresour. Technol. 98:345-352.

66. Svensson, B & Sogaard, M. 1993. Mutational analysis of glycosylase function. J. Biotechnol.
29(1):1-37.

67. Omar, N; Abou-Dobara, M & El-Sayed, A. 2011. Studies on amylase produced by some
actinomycetes.Lambert, Academic publishing Germany.

68. Ratanakhanokchai, K; Kaneko, J; Kamio, Y & Izaki, K. 1992. Purification and properties of α
maltotetraose and maltotriose producing amylase from Chloroflexus aurantiacus.
Appl.Environ. Microbiol. 58(8):2490-2494.

69. Hofemeister, B; Konig, S; Hoang, V; Engel, J; Mayer, G; Hansen, G & Hofemeister, J.1994. The
gene amyE (TV1) codes for a nonglucogenic α-amylase from Thermoactinomyces vulgaris
94-2A in Bacillus subtilis. Appl. Environ. Microbiol. 60(9):3381-3389.
70. Henrissat, B & Bairoch, A. 1993. New families in the classification of glycosyl hydrolases
based on amino acid sequence similarities. Biochem. J. 293(3):781-788.

71. Farber, G & Petsko, G.1990. The evolution of α/ß–barrel enzymes. TIBS. 15(6):228-234.

72. Machius, M; Wiegand, G & Huber, R. 1995. Crystal structure of calcium depleted Bacillus
licheniformis α-amylase at 2.2 Å resolution. J.Mol.Biol. 246(4):545-559.

73. Linden, A; Mayans, O; Meyer-Claucke, W; Antranikian, G & Wilmanns, M. 2003. Differential

regulation of a hyperthermophilic α-amylase with a novel (Ca, Zn) two metal center by zinc.
J.Biological Chemistry. 278(11):9875-9884.

http://mutagens.co.in 1898
Journal of Global Biosciences Vol. 4, SI 1, 2015 pp. 1886-1901
ISSN 2320-1355

74. Malhotra, R; Noorvez, S & Satyanarayana, T. 2000. Production and partial characterization of
thermostable and calcium-independent α-amylase of an extreme thermophile Bacillus
thermooleovorans NP54. Letters in Applied Microbiology. 31(5):378-384.

75. Bayer, E; Morag, E; Wilcheck, M; Lamed, R; Yaron, S & Shoham, Y. 1995. Cellulosome
domains for novel biotechnological application. In: Carbohydrate bioengineering,
B.Petersen, B. Srensson & S. Pedersen (Ed.). Progress in biotechnology, Elsevier. :251-259.

76. Aleshin, A; Firsov, L & Honzatko, R. 1994. Refined structure for the complex of acarbose with
glucoamylase from Aspergillus awamori Var. X100 to 2.4-A resolution. J. Biol. Chem.
269(22):15631-15639.

77. Jackson, CJ; Foo, JL; Kim, HK; Carr, PD; Liu, JW; Salem, G & Ollis, DL.2008. In crystallo capture
of a Michaelis complex and product-binding modes of a bacterial phosphotriesterase. J. Mol.
Biol. 375 :1189–1196.

78. Uitdehaag, J; Mosi, R; Kalk, K; vander Veen, B; Dijkhuizen, L; Withers, S & Dijkstra, B.1999. X-
ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate
catalysis in the α-amylase family. Nat. Struct. Biol. 6(5):432-436.

79. Crueger, W & Crueger, A. 2003. Stabilization of enzyme and cells.In:Biotechnology, A text
book of industrial microbiology II nd Ed.Panima Publishing corp, New Delhi.,:212-217.

80. Lundkvist, H & Olsen, HS. 2007. Enzyme applications in bread making. Biozoom nr. 4.
Novozymes.

81. Iqbal, J; Yousaf, M; Chaudhry, MY & Hamid, M. 1997. Designing enzyme from Bacillus subtilis
234. Pak. J. Sci. 49:1-3.
82. Allan, S; Torbenvedel, B & Henrik, BF. 1997. Recombinant alpha amylase mutants and their
use in textile desizing, starch liquification and washing. PTC. Int. Appl. 12:205-210.

83. Decordt, S; Hendrick, M; Maesmans, G & Tobback, P.1994. The influence of poly alcohols
and carbohydrate on the thermostability of alpha amylase. J. Bioeng. 43:107-114

84. Ensari, N; Birol, YO & Cetin, MA. 1996. Effect of starch induced bacterial growth and amylase
production in Bacillus subtilis. Starch/Staerk. 47:315-321.

85. Hamilton, LM; Kelly, CT & Fogarty, WM. 1998. Raw starch degradation by the non-raw
starch-absorbing bacterial alpha amylase of Bacillus sp. IMD. 434. Carbohydrates
resources. 314: 251-257.

86. Akiba, T; Kawak, YS & Kudo, T.1998. Purification and characterization of alpha-amylase
from hyper-thermophilic Thermococcus profundus, which hydrolyzes both, α1-4 and α-1-6
glucosidic linkage. J. Fermn. Bioeng. 86:363-367.

87. Shetty, JK & Crab, WD. 1990. Commodity scale production of Sugars from starches. Current
opinion in Microbiology. 2:252-256.

88. Santamaria, RIG; Delrio, G Saab; Rodriguez, ME; Soberan, X & Lpezmumguia, A.1999.
Alcoholysis reactions from starch with alpha amylases. FEBS Lett. 45: 346 -350.

89. Okolo, BN; Ezeogu, LI & Ebisike, CO.1996. Raw starch digesting amylase from
Thermoactinomyces thalophilus. F.13. J. Microb. Biotechnol. 12:637-638.

http://mutagens.co.in 1899
Journal of Global Biosciences Vol. 4, SI 1, 2015 pp. 1886-1901
ISSN 2320-1355

90. Saxena, RK; Malhotra, B & Batra, A. 2004. Commercial Importance of Some Fungal Enzymes.
In: Arora, D.K. (ed) Handbook of fungal biotechnology. Marcel Dekker, New York, USA. :
287-298.

91. Borchet, TV; Lassen, SE; Svendsen, A & Frabtzen, HB.1995. Oxidation of stable amylase for
detergent. Biotechnol. 10:175-179.

92. Atsushi, J & Eiichi, H. 1998. Carbohydrates containing laundry composition with Improved
detergency. J. Kokai Tokkyo Koho. 10:96-104.

93. Onzales, MA.1997. Laundry detergent bar composition with bleach system stabilized by
enzymes. Av. Appl. 9:110-115.

94. Mirasol, S; Takashi, E & Ritsuha, O.1997. of detergency of laundry bleach composition and
bleaching method. J. Chem. Eng. 25:1-7.

95. Ismail, AM; Omar, SH; El-Aassar, SA & Gouda, MK.1992.Purification of alpha amylase from
Bacillus lentus cultures. Appl. Microbiol. Biotechnol. 38(3):312-314.

96. Chi, Z; Liu, G; Wang, F; Ju, L & Zhang, T. 2009. Saccharomycopsis fibuligera and its
applications in biotechnology. Biotechnol Adv. 27:423-431.

97. Oner, ET. 2006. Optimization of ethanol production from starch by an amylolytic nuclear
petite Saccharomyces cerevisiae strain. Yeast. 23:849–856.

98. Moraes, LMP; Filho, SA & Ulhoa, CJ.1999. Purification and some properties of an α-amylase
glucoamylase fusion protein from Saccharomyces cerevisiae. World J. Microbiol.
Biotechnol. 15:561-564.

99. Moraes, LM; Astolfi-Filho, S & Oliver, SG.1995. Development of yeast strains for the efficient
utilisation of starch: evaluation of constructs that express alpha-amylase and glucoamylase
separately or as bifunctional fusion proteins. Appl. Microbiol Biotechnol. 43:1067-1076.

100. Sanchez , OJ & Cardona, CA.2008. Trends in biotechnological production of fuel ethanol
from different feedstocks. Bioresour Technol. 99:5270-5295.

101. Bergman, FW; Abe, J & Hizukurji, S.1988. Selection of microorganism which produce raw
starch degrading amylase. Appl. Microbiol. Biotechnol. 27:443-446

102. Friendrich, J; Cimerman, A & Perdih, A. 1987. Mixed culture of Aspergillus awamori and
Trechoderna reesi for bioconversion of apple distillery waste. Appl. Microbiol.
Biotechnol.,26,: 299-305.

103. Jamuna, R & Radhakrishna, SV.1989. SCP production and removal of organic load from
cassava starch industry by yeasts. J. Ferment.Bioengg. 67: 126-131.

104. Kingspohn, K; Bader, J; Kruse, PV & Schugerl, K. 1993. Utilization of potato pulp from
potato starch processing. Proc. Biochem. 28: 91-98.

105. Becks, S; Bielawaski, C; Henton, D; Padala, R; Burrows, K & Slaby, R.1995. Application of
a liquid stable amylase reagent on the Ciba Corning Express clinical chemistry system. Clin
Chem. 41:S186.

http://mutagens.co.in 1900
 Journal of Global Biosciences Vol. 4, SI 1, 2015 pp. 1886-1901
ISSN 2320-1355

106. Giri, NY; Mohan, AR; Rao, LV & Rao, CP.1990. Immobilization of α-amylase complex in
detection of higher oligosaccharides on paper. Curr Sci. 59:1339-1340.

107. Menzel, C; Lerch, T; Schneider, K; Weidemann, R; Tollnick, C; Kretymer, G; Scheper, T &

Schugert, K.1998. Application of biosensors with an electrolyte isolator semiconductor
capacitor (EIS-CAP)transducer for process monitoring. Process Biochem. 33:175-180.

108. Udani, J; Hardy, M & Madsen, DC. 2004.Blocking carbohydrate absorption and weight
loss: a clinical trial using Phase 2 brand proprietary fractionated white bean extract. Altern
Med Rev. 9(1):63–90.

http://mutagens.co.in 1901

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