Amylase Anti Staling

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Process Biochemistry 00 (2003) 1 /18

www.elsevier.com/locate/procbio
Microbial a-amylases: a biotechnological perspective

Rani Gupta *,1, Paresh Gigras, Harapriya Mohapatra, Vineet Kumar Goswami,
Bhavna Chauhan
Department of Microbiology, University of Delhi South Campus, Benito Juarez Marg, New Delhi 110 021, India
Received 3 July 2002; accepted 30 January 2003
Abstract
Amylases are one of the most important and oldest industrial enzymes. These comprise hydrolases, which hydrolyse starch
molecules to fine diverse products as dextrins, and progressively smaller polymers composed of glucose units. Large arrays of
amylases are involved in the complete breakdown of starch. However, a-amylases which are the most in demand hydrolyse a-1,4
glycosidic bond in the interior of the molecule. a-Amylase holds the maximum market share of enzyme sales with its major
application in the starch industry as well as its well-known usage in bakery. With the advent of new frontiers in biotechnology, the
spectrum of a-amylase application has also expanded to medicinal and analytical chemistry as well as in automatic dishwashing
detergents, textile desizing and the pulp and paper industry. Amylases are of ubiquitous occurrence, produced by plants, animals
and microorganisms. However, microbial sources are the most preferred one for large scale production. Today a large number of
microbial a-amylases are marketed with applications in different industrial sectors. This review focuses on the microbial amylases
and their application with a biotechnological perspective.
# 2003 Elsevier Science Ltd. All rights reserved.
Keywords: a-Amylase; Baking; Antistaling; Dextrinising activity; Starch liquefaction
1. Introduction
Amylases are enzymes which hydrolyse starch molecules to give diverse products including dextrins and
progressively smaller polymers composed of glucose
units [1]. These enzymes are of great significance in
present day biotechnology with applications ranging
from food, fermentation, textile to paper industries [2].
Although amylases can be derived from several sources,
including plants, animals and microorganisms, microbial enzymes generally meet industrial demands. Today
a large number of microbial amylases are available
commercially and they have almost completely replaced
chemical hydrolysis of starch in starch processing
industry [2].
The history of amylases began in 1811 when the first
starch degrading enzyme was discovered by Kirchhoff.
* Corresponding author. Tel.: /91-11-2611-1933; fax: /91-112688-5270.

E-mail address: ranigupta15@rediffmail.com (R. Gupta).
1
E-mail: microzyme@123india.com.
This was followed by several reports of digestive

amylases and malt amylases. It was much later in
1930, that Ohlsson suggested the classification of starch
digestive enzymes in malt as a- and b-amylases according to the anomeric type of sugars produced by the
enzyme reaction. a-Amylase (1,4-a-D-glucan-glucanhydrolase, EC. 3.2.1.1) is a widely distributed secretary
enzyme. a-Amylases of different origin have been
extensively studied.
Amylases can be divided into two categories, endoamylases and exoamylases. Endoamylases catalyse hydrolysis in a random manner in the interior of the starch
molecule. This action causes the formation of linear and
branched oligosaccharides of various chain lengths.
Exoamylases hydrolyse from the non-reducing end,
successively resulting in short end products. Today a
large number of enzymes are known which hydrolyse
starch molecule into different products and a combined
action of various enzymes is required to hydrolyse
starch completely.
A number of reviews exist on amylases and their
applications, however, none specifically covers a-amy-
0032-9592/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0032-9592(03)00053-0
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R. Gupta et al. / Process Biochemistry 00 (2003) 1 /18
lases at length. a-Amylases are one of the most popular

and important form of industrial amylases and the
present review highlights the various aspects of microbial a-amylases.
2. Distribution of a-amylase among microorganisms

a-Amylases are universally distributed throughout the
animal, plant and microbial kingdoms. Over the past
few decades, considerable research has been undertaken
with the extracellular a-amylase being produced by a
wide variety of microorganisms [1 /5]. The major
advantage of using microorganisms for the production
of amylases is the economical bulk production capacity
and microbes are easy to manipulate to obtain enzymes
of desired characteristics [5]. a-Amylase has been
derived from several fungi, yeasts, bacteria and actinomycetes, however, enzymes from fungal and bacterial
sources have dominated applications in industrial sectors [2].
3. Determination of a-amylase activity

a-Amylases are generally assayed using soluble starch
or modified starch as the substrate. a-Amylase catalyses
the hydrolysis of a-1,4 glycosidic linkages in starch to
produce glucose, dextrins and limit dextrins. The reaction is monitored by an increase in the reducing sugar
levels or decrease in the iodine colour of the treated
substrate. Various methods are available for the determination of a-amylase activity [6]. These are based on
decrease in starch/iodine colour intensity, increase in
reducing sugars, degradation of colour-complexed substrate and decrease in viscosity of the starch suspension.
3.1. Decrease in starch /iodine colour intensity
Starch forms a deep blue complex with iodine [7] and
with progressive hydrolysis of the starch, it changes to
red brown. Several procedures have been described for
the quantitative determination of amylase based on this
property. This method determines the dextrinising
activity of a-amylase in terms of decrease in the iodine
colour reaction.
3.1.1. Determination of dextrinising activity
The dextrinising activity of a-amylases employs
soluble starch as substrate and after terminating the
reaction with dilute HCl, iodine solution is added. The
decrease in absorbance at 620 nm is then measured
against a substrate control. One percent decline in
absorbance is considered as one unit of enzyme [8].
The major limitation of this assay is interference of
media components including Luria broth, tryptone,
peptone, corn steep liquor (CSL), etc. and thiol compounds with starch iodine complex. Copper sulphate
and hydrogen peroxide protect the starch/iodine colour
in the case of interference by these media components
[9]. Further, zinc sulphate was found to be best for
counteracting the interference of various metal ions.
Various workers [10,11] have successfully used the
original assay procedure in combination with flow
injection analysis (FIA). The flow system comprised of
an injection valve, a peristaltic pump, a photometer with
a flow cell and 570 nm filter and a pen recorder. Samples
are allowed to react with starch in a coil before iodine
was added. Absorbance is then read at 570 nm. This
method has many advantages including high sampling
rates, fast response, flexibility and simple apparatus.
3.1.2. Sandstedt Kneen and Blish (SKB) method
The SKB method [12], is one of the most widely
adopted methods for determination of amylases used in
the baking industry. The potency of most commercial
amylases is described in terms of SKB [12] units. This
method is used generally to express the diastatic strength
of the malt and not for expressing a-amylase activity
alone [13].
3.1.3. Indian pharmacopoeia method
As described in the Indian pharmacopoeia, this
method is used to calculate a-amylase activity in terms
of grams of starch digested by a given volume of enzyme
[14]. This procedure involves incubation of the enzyme
preparation in a range of dilutions in buffered starch
substrate at 40 8C for 1 h. The solutions are then treated
with iodine solution. The tube, which does not show any
blue colour, is then used to calculate activity in terms of
grams of starch digested. This method is usually
employed for estimating a-amylase activity in cereals.
3.2. Increase in reducing sugars or dinitrosalicyclic acid
(DNSA) method
This method determines the increase in reducing
sugars as a result of amylase action on starch [15]. The
major defect in this assay is a slow loss in colour
produced and destruction of glucose by constituents of
the DNSA reagent.
To overcome these limitations, a modified method for
the estimation of reducing sugars was developed [16].
Rochelle salts were excluded and 0.05% sodium sulphate
was added to prevent the oxidation of the reagent. Since
then the modified method has been used extensively to
measure reducing sugars without any further modifications in the procedure.
Alternate methods, which also rely on the estimation
of the reducing sugars are also, employed [17].
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3.3. Degradation of colour-complexed substrate

For some years, groups have been working on the
development of a specific a-amylase determination
method based on the use of new types of substrates.
These methods employ starch covalently complexed
with blue dye such as Remazol brilliant Blue R [18] or
Cibacron Blue F3 G-A [19] as an alternative substrate.
The synthesis of these substrates involves two major
steps. Soluble starch is coloured under alkaline conditions using the dye. This is the result of formation of
covalent bonds between starch and dye molecules. The
coloured starch is subsequently cross-linked by the
addition of 1,4-butanediol diglycide ether. This gives
an insoluble network, which swells in water. The
enzymic hydrolysis of such insoluble starch derivatives
yields soluble starch hydrolysates carrying the coloured
marker. This method is simple and sensitive for aamylase determination, but even minute quantities of
glucose might lead to erroneous results due to starch
contamination by dextrin substrate [19]. Recently, a
rapid and sensitive microassay based on dye cross linked
starch for a-amylase detection has been reported. It can
successfully detect as low as 0 /50 ng of enzyme [20].
Other novel substrates such as nitrophenyl derivatives
of maltosaccharides have also been employed. The assay
measures the release of free p -nitrophenyl groups. The
use of nitrophenyl-maltosaccharides in conjunction with
a specific yeast a-glucosidase can be used but these
substrates are rapidly cleaved by glucoamylases commonly present in the culture broths. The use of nonreducing end blocked p-nitrophenyl maltoheptoside
(BPNPG7) has also been described [21]. The blocking
group (4,6-O -benzylidene) prevents the hydrolysis of the
substrate by the exo-acting enzymes and is thus specific
for a-amylase. The assay is simple, reliable and accurate
but is expensive as it involves the use of a synthetic
substrate and specific enzymes. Thus the use of this
method is restricted only to very specific tests and not
for routine analysis. A comparison was made for the use
of end blocked p -nitrophenyl maltoheptoside (BPNPG7)
with a number of accepted procedures that employ
starch as the substrate. The reaction was monitored
using the starch /iodine colour [21]. There was an
excellent correlation between each of the assay procedures employed. This indicates that all the methods give
an accurate and reliable measure of a-amylase activity
and can be used as per the requirement. Both these
methods are commercially available as commercial kits,
however, it is found that a-amylases exhibit lower
affinity for low molecular weight substrates [18].
3.4. Decrease in viscosity of the starch suspension
These methods are generally used in the bakery
industry to assess the quality of the flour and not for
estimating a-amylase activity which are based on the

determination of the rheological properties of the
dough. Methods, which fall into this category, are the
falling number test and the Amylograph or Farinograph
test.
3.4.1. Falling number (FN) method
The falling number (FN) method, internationally
standardised [22 /24] is accepted for assessing cereal aamylase activity in flour /enzyme preparations at
100 8C. Both cereal and fungal a-amylases are used to
improve the fermentation of flour deficient in amylase
activities. Because fungal a-amylases have low thermostability, they cannot be detected by the standard FN
method at 100 8C [25]. This method has been modified
and standardised [25] for measuring both cereal and
fungal a-amylase activity at 300 8C, by replacing a part
of the flour with pre-gelatinised starch. A falling number
of about 400 indicates a normally malted flour.
3.4.2. Amylograph/Farinograph test
The milling and baking industries generally assess the
diastatic activity of flours by means of an amylograph.
This method is also based on the relationship of peak
viscosity of starch slurry and the enzyme activity level
[23]. The higher the enzyme activity, the thinner is the
hot paste viscosity. When the amylograph is used, values
of 400 /600 Brabender units of the Farinograph are
considered optimal for bread baking flours (higher
values indicate a lack and lower values indicate an
excess of activity).
4. Physiology of a-amylase production

The production of a-amylase by submerged fermentation (SmF) and solid state fermentation (SSF) has been
thoroughly investigated and is affected by a variety of
physicochemical factors. Most notable among these are
the composition of the growth medium, pH of the
medium, phosphate concentration, inoculum age, temperature, aeration, carbon source and nitrogen source
[5,26]. Most reports among fungi have been limited to a
few species of mesophilic fungi where attempts have
been made to specify the cultural conditions and to
select superior strains of the fungus to produce on a
commercial scale [2 /4].
4.1. Physiochemical parameters
The role of various physico-chemical parameters,
including carbon and nitrogen source, surface acting
agents, phosphate, metal ions, temperature, pH and
agitation have been studied.
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4.1.1. Substrate source: induction of a-amylase

a-Amylase is an inducible enzyme and is generally
induced in the presence of starch or its hydrolytic
product, maltose [27 /30]. Most reports available on
the induction of a-amylase in different strains of
Aspergillus oryzae suggest that the general inducer
molecule is maltose. There is a report of a 20-fold
increase in enzyme activity when maltose and starch
were used as inducers in A. oryzae (NRC 401013) [31].
Similarly strong a-amylase induction by starch and
maltose in the case of A. oryzae DSM 63303 has been
reported [29]. Apart from maltose, in some strains, other
carbon sources as lactose, trehalose, a-methyl-D-glycoside also served as inducers of a-amylase [28]. Not only
the carbon source, but also the mycelial condition/age
affect the synthesis of a-amylase by A. oryzae M-13 [28].
There are reports that 5 days starved non-growing
mycelia were the most appropriate for optimal induction
by maltose. a-Amylase production is also subjected to
catabolite repression by glucose and other sugars, like
most other inducible enzymes [30,32]. However, the role
of glucose in the production of a-amylase in certain
cases is controversial. a-Amylase production by A.
oryzae DSM 63303 was not repressed by glucose rather;
a minimal level of the enzyme was induced in its
presence [29]. However, xylose or fructose have been
classified as strongly repressive although they supported
good growth in Aspergillus nidulans [33].
The carbon sources as glucose and maltose have been
utilised for the production of a-amylase. However, the
use of starch remains promising and ubiquitous. A
number of other non-conventional substrates as lactose
[34], casitone [35,36], fructose [37], oilseed cakes [38] and
starch processing waste water [39] have also been used
for the production of a-amylase while the agro-processing byproduct, wheat bran has been used for the
economic production of a-amylase by SSF [5]. The use
of wheat bran in liquid surface fermentation (LSF) for
the production of a-amylase from Aspergillus fumigatus
and from Clavatia gigantea, respectively, has also been
reported [40,41]. High a-amylase activities from A.
fumigatus have also been reported using a-methyl-Dglycoside (a synthetic analogue of maltose) as substrate
[42].
Use of low molecular weight dextran in combination
with either Tween 80 or Triton X-100 for a-amylase
production in the thermophilic fungus Thermomyces
lanuginosus (ATCC 200065) has been reported [43].
Triton X-100 had no effect, whereas Tween 80 increases
the a-amylase activity 27-fold.
4.1.2. Nitrogen sources
Organic nitrogen sources have been preferred for the
production of a-amylase. Yeast extract has been used in
the production of a-amylase from Streptomyces sp. [44],
Bacillus sp. IMD 435 [45] and Halomonas meridiana
[46]. Yeast extract has also been used in conjunction

with other nitrogen sources such as bactopeptone in the
case of Bacillus sp. IMD 434 [47], ammonium sulphate
in the case of Bacillus subtilis [48], ammonium sulphate
and casein for C. gigantea [40] and soybean flour and
meat extract for A. oryzae [49]. Yeast extract increased
the productivity of a-amylase by 110/156% in A. oryzae
when used as an additional nitrogen source than when
ammonia was used as a sole source [50]. Various other
organic nitrogen sources have also been reported to
support maximum a-amylase production by various
bacteria and fungi. However, organic nitrogen sources
viz. beef extract, peptone and com steep liquor supported maximum a-amylase production by bacterial
strains [35,38,51 /54] soybean meal and casamino acids
by A. oryzae [55]. CSL has also been used for the
economical and efficient production of a-amylase from
a mutant of B. subtilis [56]. Apart from this, various
inorganic salts such as ammonium sulphate for A.
oryzae [30] and A. nidulans [29], ammonium nitrate
for A. oryzae [57] and Vogel salts for A. fumigatus [42]
have been reported to support better a-amylase production in fungi.
Amino acids in conjunction with vitamins have also
been reported to affect a-amylase production. However,
no conclusion can be drawn about the role of amino
acids and vitamins in enhancing the a-amylase production in different microorganisms as the reports are
highly variable. a-Amylase production by Bacillus
amyloliquefaciens ATCC 23350 increased by a factor
of 300 in the presence of glycine [58]. The effect of
glycine was not only as a nitrogen source rather it
affected a-amylase production by controlling pH and
subsequently amylase production increased. b-Alanine,
DL-nor valine and D-methionine were effective for the
production of alkaline amylase by Bacillus sp. A-40-2.
However, the role of amino compounds was considered
to be neither as nitrogen nor as a carbon source, but as
stimulators of amylase synthesis and excretion [59]. It
has been reported that only asparagine gave good
enzyme yields [57] while the importance of arginine for
a-amylase production from B. subtilis has also been well
documented [60].
4.1.3. Role of phosphate

Phosphate plays an important regulatory role in the
synthesis of primary and secondary metabolites in
microorganisms [61,62] and likewise it affects the growth
of the organism and production of a-amylase. A
significant increase in enzyme production and conidiation in A. oryzae above 0.2 M phosphate levels has been
reported [55]. Similar findings were corroborated in B.
amyloliquefaciens where low levels of phosphate resulted
in severely low cell density and no a-amylase production
[63]. In contrast, high phosphate concentrations were
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inhibitory to enzyme production by B. amyloliquefaciens

[58].
4.1.4. Role of other ions
K , Na , Fe2, Mn2, Mo2, Cl , SO2
had no
4
effect while Ca2 was inhibitory to amylase production
by A. oryzae EI 212 [57]. Mg2 played an important
role and production was reduced to 50% when Mg2
was omitted from the medium. Na and Mg2 show
coordinated stimulation of enzyme production by Bacillus sp. CRP strain [64]. Addition of zeolites to control
ammonium ions in B. amyloliquefaciens resulted in
increased yield of a-amylase [65]. An inverse relationship between a-amylase production and growth rate was
observed for Streptomyces sp. in the presence and
absence of Co2 [66], the presence of Co2 enhancing
the final biomass levels by 13-fold, albeit with a
reduction in enzyme yield.
4.1.5. pH
Among the physical parameters, the pH of the growth
medium plays an important role by inducing morphological change in the organism and in enzyme secretion.
The pH change observed during the growth of the
organism also affects product stability in the medium.
Most of the Bacillus strains used commercially for the
production of bacterial a-amylases by SmF have an
optimum pH between 6.0 and 7.0 for growth and
enzyme production. This is also true of strains used in
the production of the enzyme by SSF. In most cases the
pH used is not specified excepting pH 3.2 /4.2 in the case
of A. oryzae DAE 1679 [39], 7.0 /8.0 in A. oryzae EI 212
[57] and 6.8 for B. amyloliquefaciens MIR-41 [67]. In
fungal processes, the buffering capacity of some media
constituents sometimes eliminates the need for pH
control [68]. The pH values also serves as a valuable
indicator of the initiation and end of enzyme synthesis
[69]. It is reported that A. oryzae 557 accumulated aamylase in the mycelia when grown in phosphate or
sulphate deficient medium and was released when the
mycelia were replaced in a medium with alkaline pH
(above 7.2) [28].
4.1.6. Temperature
The influence of temperature on amylase production
is related to the growth of the organism. Among the
fungi, most amylase production studies have been done
with mesophilic fungi within the temperature range of
25 /37 8C. Optimum yields of a-amylase were achieved
at 30 /37 8C for A. oryzae [55,57]. a-Amylase production has also been reported at 55 8C by the thermophilic
fungus Thermomonospora fusca [70] and at 50 8C by T.
lanuginosus [17].
a-Amylase has been produced at a much wider range
of temperature among the bacteria. Continuous production of amylase from B. amyloliquefaciens at 36 8C has
been reported [67]. However, temperatures as high as

80 8C have been used for amylase production from the
hyperthermophile Thermococcus profundus [71].
4.1.7. Agitation
Agitation intensity influences the mixing and oxygen
transfer rates in many fungal fermentations and thus
influences mycelial morphology and product formation
[69,72/76]. It has been reported that a higher agitation
speed is sometimes detrimental to mycelial growth and
thus may decrease enzyme production. However, it is
reported that the variations in mycelial morphology as a
consequence of changes in agitation rate do not affect
enzyme production at a constant specific growth rate
[76].
Agitation intensities of up to 300 rpm have normally
been employed for the production of amylase from
various microorganisms as reported in the literature.
5. Fermentation studies on a-amylase production

The effect of environmental conditions on the regulation of extracellular enzymes in batch cultures is well
documented [77]. A lot of work on the morphology and
physiology of a-amylase production by A. oryzae during
batch cultivation has been done. Accordingly, morphology of A. oryzae was critically affected by the growth
pH [78]. In a series of batch experiments, authors
observed that at pH 3.0 /3.5, freely dispersed hyphal
elements were formed. In the pH range 4/5, both pellets
and freely dispersed hyphal fragments were observed
whereas at pH higher than 6 pellets were the only
growth forms recorded. Other groups [39,79] have
recorded similar observations for other strains of
A. oryzae . The optimum growth temperature was found
to be 35 8C. It is demonstrated that when glucose was
exhausted the biomass production stopped whereas the
secretion of a-amylase increased rapidly [79]. One report
states that inoculum quantity did not affect morphological changes in A. oryzae in air-lift bioreactors and that
pellet size decreased considerably as the air velocity
increased [39]. In the case of a-amylase production by
Bacillus flavothermus in batch cultivation in a 20 l
fermentor, a-amylase production and biomass peaked
twice and highest activity was obtained after 24 h [34]. It
was observed that the kinetics of enzyme synthesis was
more of the growth associated than non-growth associated type [35]. Similar findings were cited in another
report with B. amyloliquefaciens [63].
Continuous and fed-batch cultures have been recognised as most effective for the production of the enzyme
[60]and several groups have studied the effectiveness of
these cultures. The production of a-amylase from
B. subtilis TN106 (pAT5) was enhanced substantially
by extending batch cultivation with fed-batch operation
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[60]. The bulk enzyme activity was nearly 54% greater in

a two-stage fed-batch operation at a feed rate of 31.65
ml h 1 of medium, than that attained in the single stage
batch culture. The effects of controlled feeding of
maltose at a feed rate of 1 /4 g h1 for a-amylase and
glucoamylase production from A. oryzae RIB 642 in a
rotary draft tube fermentor (RTF) have been studied
[49]. At a feed rate of 1 g h1 the yields of a-amylase
were twice than those obtained in batch cultures. When
fed-batch cultivations were performed on a pilot scale
RTF at a feed rate of 24 g h1, the biomass and aamylase yields was higher than those obtained in a
laboratory scale jar fermentor.
A model to simulate the steady-state values for
biomass yield, residual sugar concentration and specific
rate of a-amylase production has been proposed which
simulated experimental data very well [80]. Furthermore, it was found in chemostat experiments that the
specific rate of a-amylase production decreased by up to
70% with increasing biomass concentration at a given
dilution rate. Shifts in the dilution rate in continuous
culture could be used to obtain different proportions of
the enzymes, by the same strain [66]. It was further
demonstrated that maximum production of a-amylase
occurred in continuous culture at a dilution rate of 0.15
h1 and amylase activity in the culture was low at
dilution rates above 1.2 h 1. In contrast, in Bacillus sp.
the switching of growth from batch to continuous
cultivation resulted in the selection of a non a-amylase
producing variant [63]. A decline in enzyme production
was also accompanied by morphological and metabolic
variations during continuous cultivation [81,82].
The industrial exploitation of SSF for enzyme production has been confined to processes involving fungi
and it is generally believed that these techniques are not
suitable for bacterial cultivation [5]. The use of SSF
technique in a-amylase production and its specific
advantages over other methods has been discussed
extensively [5].
6. Purification of microbial a-amylases

Industrial enzymes produced in bulk generally require
little downstream processing and hence are relatively
crude preparations. The commercial use of a-amylase
generally does not require purification of the enzyme,
but enzyme applications in pharmaceutical and clinical
sectors require high purity amylases. The enzyme in
purified form is also a prerequisite in studies of
structure /function relationships and biochemical properties.
The purification of a-amylases from microbial sources
in most cases has involved classical purification methods. These methods involve separation of the culture
from the fermentation broth, selective concentration by
precipitation using ammonium sulphate or organic

solvents such as chilled acetone. The crude enzyme is
then subjected to chromatography, usually affinity, ion
exchange and/or gel filtration. A number of reviews are
available on purification and characterisation of aamylases from a range of microorganisms [1,2,4,26,83].
Table 1 summarises various purification strategies
adopted for microbial a-amylases.
7. Biochemical properties of a-amylases

The enzymic and physicochemical properties of aamylases from several microorganisms have been extensively studied and described [2 /4,83]. A summary is
presented in Table 2.
7.1. Substrate specificity
As holds true for the other enzymes, the substrate
specificity of a-amylase varies from microorganism to
microorganism. In general, a-amylases display highest
specificity towards starch followed by amylose, amylopectin, cyclodextrin, glycogen and maltotriose.
7.2. pH optima and stability
The pH optima of a-amylases vary from 2 to 12 [4]. aAmylases from most bacteria and fungi have pH optima
in the acidic to neutral range [2]. a-Amylase from
Alicyclobacillus acidocaldarius showed an acidic pH
optima of 3 [84], in contrast to the alkaline amylase
with optima of pH 9 /10.5 reported from an alkalophilic
Bacillus sp. [85 /88]. Extremely alkalophilic a-amylase
with pH optima of 11 /12 has been reported from
Bacillus sp. GM8901 [89]. In some cases, the pH
optimum was observed to be dependent upon temperature as in the case of Bacillus stearothermophilus DONK
BS-1 [90] and on calcium as in the case of B.
stearothermophilus [91].
a-Amylases are generally stable over a wide range of
pH from 4 to 11 [3,4,45,47,85,92], however, a-amylases
with stability in a narrow range have also been reported
[46,86,93].
7.3. Temperature optima and stability
The temperature optimum for the activity of aamylase is related to the growth of the microorganism
[4]. The lowest temperature optimum is reported to be
25 /30 8C for F. oxysporum amylase [94] and the highest
of 100 and 130 8C from archaebacteria, Pyrococcus
furiosus and Pyrococcus woesei , respectively [95 /97].
Temperature optima of enzymes from Micrococcus
varians are calcium dependent [98] and that from H.
meridiana is sodium chloride dependent [46].
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Table 1
Purification strategies employed for a-amylase
Microorganism
Fungi and yeast

A. oryzae NRC 401013
A. flavus LINK
Cryptococcus sp. S-2
L. kononenkoae CBS5608
Saccharomyces cerevisiae
YPB-G
Schwanniomyces alluvius
UCD-54-83
Thermomonospora curvata
T. lanuginosus
T. lanuginosus IISc91
Bacteria
Bacillus sp. IMD435
Bacillus sp. IMD 434
Bacillus sp. WN 11
B. licheniformis CUMC 305
B. licheniformis NCIB 6346
B. stearothermophilus ATCC
12980
B. subtilis
B. subtilis
B. subtilis 65
Lactobacillus plantarum A6
Pseudomonas stutzeri
Streptococcus bovis JB1
Thermomonospora curvata
NCIMB 10081
T. profundus DT5432
Purification strategy
Fold purification/
yield (%)
DE52-Cellulose (pH 7.0), 70% (NH4)2SO4, Sephacryl S300, 70% (NH4)2SO4,

DE52-Cellulose (pH 7.0)
50 /90% (NH4)2SO4, DEAE-Sephadex A50 (pH 6.5)
Ultrafiltration, a-Cyclodextrin coupled with Sepharose 6B (pH 7.0)
60% (NH4)2SO4, crosslinked starch (pH 8.5), DEAE Bio-Gel A (pH 5.5)
Ultrafiltration, b-Cyclodextrin linked Sepharose 6B (Epoxy activated, pH 4.5),
Sephadex G-100 (pH 4.5)
Ultrafiltration, DEAE-sephacel (pH 5.6), Sephadex G-150 (pH 5.6)
Reference
[31]
13.8/70
140/78
6000/52
5/2
[92]
[152]
[99]
[153]
10.8/17.1
[154]
Ultrafiltration, 75% ethanol precipitation, Sephadex G-150 (pH 8.0), DEAE

66/9
Cellulose, ultrafiltration
Ultrafiltration, DEAE-Trisacryl (pH 7.0), Phenyl-Sepharose (pH 7.0)
Ultrafiltration, DEAE-Sephadex A50 (pH 5.0), ultrogel AcA54, DEAE-Sephadex 112/41
A50 (pH 8.0), Bio-Gel P-30
[155]
a-Cyclodextrin coupled Sepharose 6B (pH 6.0)

Acetone precipitation, Resource Q (pH 7.0), Phenyl Sepharose CL-4B (pH 7.8)
60% (NH4)2SO4, DEAE Sepharose (pH 5.3), Sephadex G-75
744/65
266/ /
Amy I 65/13, Amy II
40.7/9.5
212/42
33/66
/
[45]
[47]
[100]
9/17
2.5/ /
30.85/24.8
20/35
1.036/ /
6.9/50
300/ /
[159]
[83]
[51]
[160]
[93]
[161]
[162]
65% (NH4)2S04, CM-Cellulose (pH 6.4)

DEAE-Cellulose DE52 (pH 5.3)
Adsorption on soluble starch (1%) in 10% (NH4)2SO4, washing with Aces (pH
7.5) and 10% (NH4)2SO4, DEAE chromatography (Zetaprep disk), ultrafiltration
60% (NH4)2SO4, Sephacryl-S200 HR (pH 8.0), 60% (NH4)2SO4, S-Sepharose
Ultrafiltration
Sephacryl S-300, CM Sephadex C-50
Ultrafiltration, 50 /80% (NH4)2SO4, ultrafiltration, DEAE-Cellulose
Concentrated by drum humidifier, 25% (NH4)2SO4, 70% acetone
70% (NH4)2SO4, Sephadex G-25 (pH 7.5), Mono Q
85% (NH4)2S04, ultrafiltration, gel filtration (pH 6.0), DEAE-Sephacel (pH 8.O)
80% (NH4)2SO4, DEAE-Toyopearl 650 M (pH 7.5), Superdex 200 HR (pH 7.5) 816/26
Thermostabilities have not been estimated defactor in

many studies. Thermostabilities as high as 4 h at 100 8C
have been reported for Bacillus licheniformis CUMC
305 [86]. Many factors affect thermostability. These
include the presence of calcium, substrate and other
stabilisers [4]. The stabilising effect of starch was
observed in a-amylases from B. licheniformis CUMC
305 [85], Lipomyces kononenkoae [98] and Bacillus sp.
WN 11 [100]. Thermal stabilisation of the enzyme in the
presence of calcium has also been reported from time to
time [100 /102].
7.4. Molecular weight
Molecular weights of a-amylases vary from about 10
to 210 kDa. The lowest value, 10 kDa for Bacillus
caldolyticus [103] and the highest of 210 kDa for
Chloroflexus aurantiacus has been reported [104]. Molecular weights of microbial a-amylases are usually 50/
60 kDa as shown directly by analysis of cloned aamylase genes and deduced amino acid sequences [4].
[156]
[17]
[86]
[157]
[158]
[71]
Carbohydrate moieties raise the molecular weight of

some a-amylases. Glycoproteins have been detected in
A. oryzae [105,106], L . kononenkoae [98], B. stearothermophilus [107] and B. subtilis strains [108,109]. Glycosylation of bacterial proteins is rare. A carbohydrate
content as high as 56% has been reported in S. castelii
[110] whereas this is about 10% for other a-amylases [4].
7.5. Inhibitors
Many metal cations, especially heavy metal ions,
sulphydryl group reagents, N -bromosuccinimide, p hydroxyl mercuribenzoic acid, iodoacetate, BSA,
EDTA and EGTA inhibit a-amylases.
7.6. Calcium and stability of a-amylase
a-Amylase is a metalloenzyme, which contains at least
one Ca2 ion [111]. The affinity of Ca2 to a-amylase is
much stronger than that of other ions. The amount of
bound calcium varies from one to ten. Crystalline Taka-
Table 2
Properties of some microbial amylases
Molecular
weight
(kDa)
pH optima/stabi- Temperature oplity

tima/stability
Inhibitors
Stabilisers
Additional properties
Reference
Fungi and yeast

A. oryzae
/
/
65.4/5.0 /9.0
/
/
Km (0.13%)
[28]
A. flavus LINK
3.5
52.5
6.0/6.0 /10.0
Ag2 , Hg2
Ca2
A. foetidus ATCC
10254
A. awamori
/
41.5
5.0/ /
(60
/
/
Km (0.5 g l 1); Vmax (108.67 [92]

mM reducing sugar mg1
protein min 1
Km /2.19 mg ml 1
[163]
/
/
5.0/6.0 /7.0
(10
Ag , Cu2 , Fe3 , Hg2 , halides Substrate
A. awamori ATCC
22342
A. chevalieri NSPRI
105
A. flavus
4.2
54.0
(60
Hg2 , Pb2 , maltose
/
68.0
4.8 /5.0/3.5 /6.5

(24 h)
5.5/ /
(15
EDTA, DNP
Ca2 , Mg2
/
/
5.25/5.0 /8.0
(10
Ag , Cu2 , Hg2 , halides
Substrate
A. fumigatus
/
/
6.0/ /
(40
/
/
/
[41]
A. hennebergi Blochweitz
A. niger
/
50.0
5.5/ /
(15
/
/
/
[166]
3.44
58.0
4.0 /5.0/2.2 /7.0
min)
/
Ca2
Acid stable
[167 /
171]
3.75
A. niger ATCC 13469 /
A. niger van Tieghem /
CFTRI 1105
61.0
/
56.23
//40 8C (15 min)

50 8C/ B/60 8C
60 8C/65 8C (10
min)
/
/
Ag , Al3 , Cu2 , Hg2 , Pb2 ,
Zn2 , EDTA
Ca2
/
Ca2
/
NaF and MgSO4 stimulation
[172]
[173 /
175]
A. oryzae
/
/
5.0 /6.0/5.0 /8.5

5.0/4.0 /6.0
5.0; 6.0/5.2 /6.0
(/Ca); 5.8 /7.0
(/Ca)
5.0/6.0 /8.0
Ag , Cu2 , Fe3 , Hg2 , halides Substrate
/
[164]
A. oryzae
A. oryzae
/
/
/
53.0
40 8C/55 8C (10
min)
35 /37 8C/ /
//60 8C (90 min,
/Ca) 50 8C (30
min, /Ca)
/
PCMB
/
Ca2
Km /7.13, 4.35, 3.12 mM

Taka Diastase, TakaAmylase A, Km / 29,
2.4%, 4.7, 10.2, 2.4 mM
[176]
[177 /
180]
30 /40 8C/ /
/
/
Km /4.16 mg ml 1
[181,182]
Higher thermal stability

than commercial Takaamylase
Km (0.13%)
[183]
50 8C/50 8C (30
min)
55 8C/50 8C (1 h)
45 8C/35 8C
min)
40 8C/55 8C
min)
50 8C/40 8C
min)
40 8C/60 8C
min)
50 8C/55 8C
min)
50 8C/60 8C
min)
50 8C/40 8C
min)
//60 8C (15
A. oryzae 245 (ATCC /

9376)
A. usamii
/
/
4.8 /6.6/ /
5.0 /5.9/5.8 /7.2
(over a year,
10 8C); 5.0 /8.2
(37 8C, 30 min)
5.0 /6.0/ /
54.0
3.0 /5.5/ /
60 /70 8C/ /
/
/
A. oryzae M13
52.0
5.4/5.0 /9.0
50 8C/ 5/50 8C
(min)
/
/
4.0
[164]
Km /1 mg ml 1
[32]
Km /0.19 mg ml 1
[165]
[164]
[28]
ARTICLE IN PRESS
pI
Source
Table 2 (Continued )
Source
pI
Molecular
weight
(kDa)

tima/stability
Inhibitors
Stabilisers
Cryptococcus S-2
4.2
66.0
6.0/ /
Fusarium vasinfectum
Atk
L. kononenkoae CBS
5608
/
/
3.5
76.0
/
50 /60 8C/90 8C
(CaCl2)
Hg2 , Ag2 , Cu2 , Zn2
/
4.4 /5.0:5.8:7.8 /
8.0/3.8 /10.0
4.5 /5.0/5.0 /7.0
(1 h)
45 /50 8C/50 8C
(30 min)
70 8C/ /
Cu2 , Mn2 , Zn2
/
Raw starch digesting en[152]

zyme; end products, G1, G2,
G3, G4
/
[184]
DTT, Cu2 , Ag2
Starch
69.0
4.0/4.0 /9.0
/
/
54.1
5.0/ /
45 8C/45 8C (10
min, /Ca)
50 8C/ /
[99]
Ca2
Km (0.8 g l 1); Kcat (622

s 1); insensitive to Ca2 ;
end products, G3, G4, G5,
G6
/
/
/
End products, G1, G2, G3
[153]
Km (0.364 mg ml 1); end

[154]
product, G1
A.E. (44 kJ mol 1; Km (2.5 [17]
mg ml 1); end product, G2
/
[186]
61.9
6.3/4.5 /7.5
40 8C/ 5/40 8C
/
/
/
42.0
5.6/ /
/
Ca2
Trichoderma viride
/
/
5.0 /5.5/4.0 /7.0
65 8C/50 8C ( /7
h)
//60 8C (10 min)
/
/
/
22.5
28.0
6.0/4.5 /9.0
9.0/6.0 /11.0
9.0/7.0 /9.0
45 /55 8C/ /
76 8C/ B/60 8C
90 8C/60 8C (3 h),
100 8C (4 h) in
presence of soluble starch
/
/
Hg2 , Cu2 , Ni2 , Zn2 , Ag2 ,
Fe2 , Co2 , Cd2 , Al3 , Mn2 ,
p- chloromercuribenzoic acid, sodium iodoacetate, EDTA
B. licheniformis NCIB /
6346
B. stearothermophilus 4.82
62 /65
7.0/7.0 /10.0
/
/
4.6 /5.1/ /
70 /90 8C/85 8C
(1 h)
55 /70 8C/ /
/
/
Na2 , Ca2 Mg2 , azide, F ,
2
2
2
SO2
3 , SO4 , S2O3 , MoO4 ,
2
WO4 , cysteine, glutathione,
thiourea, b-mercaptoethanol,
sod. glycerophosphate
/
EDTA
Ca2
B. stearothermophilus
ATCC 12980
8.8
59.0
5.0 /6.0/6.0 /7.5

(1 h, 80 8C)
B. stearothermophilus
MFF4
B. subtilis
/
/
5.5 /6.0/ /
/
48.0
6.5/5/7.0
B. subtilis 65
/
68.0
6.0/6.0 /9.0
70 /80 8C/(5 days)

70 8C or (45 min)
90 8C
70 /75 8C/half life
5.1 h at 80 8C
50 8C/ 5/50 8C
Cd2 , Cu2 , Hg2 , Pb2 , Zn2 , Ca2 , Na2 , B.S.A.

denaturation by 6 M urea
60 8C/60 8C (5
min)
Cu2 , Fe3 , Mn2 , Hg2 , Zn2 , Ca2

Pb2 , Al3 , Cd2 , Ag2 , EDTA
/
/
Hg2 , Fe3 , Al3
Mn2 , Co2
/
End product, G5
E.A. (5.1/105 J mol 1);
Km (1.274 mg ml 1); Vmax
(0.738 mg glucose ml 1
min1
[187]
[85]
[86]
End products, G1, G2, G3,

G5
Higher affinity for branched
chain substrate; E.A. (14
kcal); extremely resistant to
heat inactivation; effect of
EDTA reversed by Ca2
Km /14 mg ml 1; enzyme
active after acetone and
ethanol treatment
Ca2 enhances thermostability
Km (3.845 mg ml 1); Vmax
(585.1 mg); end product, G2
End products, G1, G2
[157]
[101]
[4]
[102]
[159]
[51]
ARTICLE IN PRESS
/
[185]
Paecilomyces sp.
ATCC 46889
Saccharomyces cerevisiae
Schwanniomyces alluvius UCD 5483
T. lanuginosus IISc 91
Bacteria
B. brevis HPD 31
/
B. licheniformis
/
B. licheniformis
/
CUMC 305 licheniformis CUMC 305
Reference
10
Source
pI
Molecular
weight
(kDa)

tima/stability
Inhibitors
Stabilisers
Reference
B. licheniformis M27
/
56.0
/
Ca2
5.6
63.0
/
/
5.9
69.2
6.0/4.0 /9.0
65 8C/40 8C (1 h)
N -Bromosuccinimide, p -hydroxymercuribenzoic acid
Cysteine, DTT
Bacillus sp. US 100
/
/
5.6/4.5 /8.0
82 8C/90 /95 8C
/
Starch, Ca2
Bacillus sp. WN 11
/
/
5.0 /8.0/ /
75 /80 8C/ /
/
/
Bacillus sp. WN 11
/
5.5/5.5 /9.0 (1 h) 75 /80 8C/80 8C

(4 h)
Fe3 , Hg2 , Cu2
/
Bacillus sp. XAL 601
/
Amy 176.0, Amy

2-53.0
/
E.A. (25 kJ mol 1); thermostability dependent upon

pH stability
G4
End products, G1, G2; specificity for raw starch; Km (
1.9 mm)
Half-life increases to 110 8C
in presence of 20% (w/v)
substrate
No requirement for Ca2 ;
starch increases temperature
stability
G4
[188]
6.5 /7.0 and 8.5 / 85 /90 8C/ /

9.0/5/7.0 and ]/ 90 8C
7.5
6.0 and 6.5
/
9.0/ /
70 8C/ /
/
/
/
48.0
/
6.5/5/7.0
7.0/5.0 /7.0
50 8C/ B/70 8C
37 8C/ /
Hg2 , Fe3 , Al3

/
Mn2 , Co2
Ca2
/
50.0
5.5//3.0 / B/8.0 65 8C/ /
Micromonospora melanosporea
M. melanosporea
Pseudomonas stutzeri
7.6
45.0
7.0/ /
7.6
/
45.0
12.5
7.0/6.0 /12.0
8.0/7.0 /9.5
55 8C/40 8C (pH
11 /12, 40 min)
55 8C/ /
47 8C/40 8C (1 h)
Streptococcus bovis
JB1
4.5
77.0
5.0 /6.0/5.5 /8.5
Streptomyces sp. IMD 8.9(1),

2679
8.7(2),
7.2(3)
T. profundus DT5432 /
47.8
Thermomonospora
curvata
[189]
[100]
[190]
[87]
[159]
[46]
[160]
/
[191]
End product, G1
E.A. (13 400 and 5200 cal
mol 1; end product, G4
Km (0.88 mg ml 1); Kcat
(2510 mmol reducing sugar
mg1 protein); end products, G2, G3, G4
End products, G1, G3; Km
(8.0 /8.2 mM)
[191]
[93]
Ca2
End products, G2, G3; Km

(0.23%)
[71]
/
End product, G2; low affinity for G3
[162]
/
/
/
Ca2
//50 8C (1 h)
Hg2 , p -chloromercuribenzoic
acid (both reversible by DTT)
/
5.5/ /
60 8C/ /, 60 /
65 8C/ /, 65 8C/ /
/
/
42.0
5.5 /6.0/5.9 /9.8
60.9
6.0/ /
80 8C/80 8C (3 h), Iodoacetic acid, N -bromosuccinic

90 8C (15 min)
acid, SDS, guanidine hydrochloride
65 8C/ /
/
[161]
[44]
ARTICLE IN PRESS
6.2
/
N -bromosuccinimide, iodine,
acetic acid, Hg2 , dimethyl aminobenzaldehyde
/
/
[47]
Escherichia coli
H. meridiana DSM
5425
L. plantarum A6
Adsorbs to raw starch or

cellulose hydrolysis products, G2 and G4
/
End products, G2, G3;
showed activity in 30% salts
Km (2.38 g l 1); A.E. (30.9
kJ mol 1)
[45]
ARTICLE IN PRESS
Starch, Ca2
/
/
/
T. fusca YX
G1, glucose; G2, maltose; G3, maltotriose; G4, maltotetraose; G5, maltopentaose; E.A., enzyme activation energy; kcal, kilo calories; kJ, kilo joules.
End products, G4, G5; Km [155]

(0.3 mg ml 1)
End products, G3, G4, G6; [70]
Km (3.3 mg ml 1); E.A. (59
kJ mol 1)
B.S.A.
5.5 /6.0/activated 65 8C/ /

at pH 7.0 /8.0
6.0/ /
60 8C/ B/65 8C
/
T. curvata
62.0
Inhibitors
tima/stability
Molecular
weight
(kDa)
pI
Source
/
Stabilisers
Reference
11
amylase A (TAA) contains ten Ca2 ions but only one

is tightly bound [112]. In other systems usually one
Ca2 ion is sufficient to stabilise the enzyme. Ca2 can
be removed from amylases by dialysis against EDTA or
by electrodialysis. Calcium free enzymes can be reactivated by adding Ca2 ions. Some studies have been
carried out on the ability of other ions to replace Ca2
as Sr2 in B. caldolyticus amylase [113]. Ca2 in TAA
has been substituted by Sr2 and Mg2 in successive
crystallisation in the absence of Ca2 and in excess of
Sr2 and Mg2 [114]. EDTA inactivated TAA can be
reactivated by Sr2, Mg2 and Ba2 [114]. In the
presence of Ca2, a-amylases are much more thermostable than without it [4,115]. a-Amylase from A. oryzae
EI 212 is inactivated in the presence of Ca2, but retains
activity after EDTA treatment [116]. There are also
reports where Ca2 did not have any effect on the
enzyme [117].
8. Industrial applications of a-amylase

Amylases are among the most important hydrolytic
enzymes for all starch based industries, and the commercialisation of amylases is oldest with first use in
1984, as a pharmaceutical aid for the treatment of
digestive disorders. In the present day scenario, amylases find application in all the industrial processes such
as in food, detergents, textiles and in paper industry, for
the hydrolysis of starch. In this light, microbial amylases
have completely replaced chemical hydrolysis in the
starch processing industry. They can also be of potential
use in the pharmaceutical and fine chemical industries.
Today, amylases have the major world market share of
enzymes [118]. Several different amylase preparations
are available with various enzyme manufacturers for
specific use in varied industries. A comprehensive
account on commercial applications of a-amylases is
quoted by Godfrey and West [119]. Various applications
of a-amylase are dealt here in brief.
8.1. Bread and baking industry and as an antistaling
agent
The baking industry has made use of these enzymes
for hundreds of years to manufacture a wide variety of
high quality products. For decades, enzymes such as
malt and microbial a-amylases have been widely used in
the baking industry [120,121]. These enzymes were used
in bread and rolls to give these products a higher
volume, better colour and a softer crumb. It is the
malt preparation that has led the way and opened the
opportunities for many enzymes to be used commercially in baking. Today, many enzyme preparations such
as proteases, lipases, xylanases, pullulanases, pentosanases, cellullases, glucose oxidases, lipoxygenases etc.
ARTICLE IN PRESS
12
are being used in the bread industry for varied purposes

[13,99,121 /123], but none had been able to replace aamylases.
Till date, the a-amylases used in baking have been
cereal enzymes from barley malt and microbial enzymes
from fungi and bacteria [124,125]. Fungal a-amylases
have been permitted as bread additives since 1955 in the
US and in 1963 in UK after confirmation of their GRAS
status [126]. Presently they are used all over the world to
different extents. Supplementation of flour with exogenous fungal a-amylase having higher activities is
common in the present day modern and continuous
baking process [126]. a-Amylase supplementation in
flour not only enhances the rate of fermentation and
reduces the viscosity of dough (resulting in improvements in the volume and texture of the product, but also
generates additional sugar in the dough, which improves
the taste, crust colour and toasting qualities of the bread
[127]. One of the new applications of a-amylase in the
industry has been in retarding the staling of baked
products, which reduces the shelf life of these products.
Upon storage the crumb becomes dry and firm, the
crust loses its crispness and the flavour of the bread
deteriorates. All these undesirable changes in the bread
are together known as staling. The importance of
retrogradation of starch fraction in bread staling has
been emphasised [128]. A loss of more than US $1
billion is incurred in USA alone every year due to the
staling of bread.
Conventionally various additives are used to prevent
staling and improve the texture and flavour of baked
products. Additives include chemicals, small sugars,
enzymes/their combinations, milk powder; emulsifiers,
monoglycerides/diglycerides, sugar esters, lecithin, etc;
granulated fat, anti-oxidant (ascorbic acid or potassium
borate), sugars/salts [129]. Recently emphasis has
been given to the use of enzymes in dough improvement/as anti-staling agents, e.g. a-amylase [130,131],
branching enzymes [132] and debranching enzymes
[133], maltogenic amylases [134], b-amylases [135]
amyloglucosidases [136]. Pullulanases and a-amylase
combination are used for efficient antistaling property
[133]. However, a slight excess of a-amylases was also
used which is undesirable as it causes stickiness in bread
[134]. Therefore, a recent trend is to use intermediate
temperature stable (ITS) a-amylases [13,124,125,137].
They are active after starch gelatinisation and become
inactive much before the completion of the baking
process. Further, the dextrin with 4/9 degree of polymerisation produced by these shows the anti-staling
properties. Although a wide variety of microbial aamylases is known, a-amylase with ITS property has
been reported from only a few microorganisms
[99,123,138,139].
8.2. Starch liquefaction and saccharification

The major market for a-amylases lies in the production of starch hydrolysates such as glucose and fructose.
Starch is converted into high fructose corn syrups
(HFCS). Because of their high sweetening property,
these are used in huge quantities in the beverage
industry as sweeteners for soft drinks. The process
requires the use of a highly thermostable a-amylase for
starch liquefaction. The use of enzyme in starch
liquefaction is well established and has been extensively
reviewed [2,140].
8.3. Textile desizing
Modern production processes for textiles introduce a
considerable strain on the warp during weaving. The
yarn must, therefore, be prevented from breaking. For
this purpose a removable protective layer is applied to
the threads. The materials that are used for this size
layer are quite different. Starch is a very attractive size,
because it is cheap, easily available in most regions of
the world, and it can be removed quite easily. Good
desizing of starch sized textiles is achieved by the
application of a-amylases, which selectively remove the
size and do not attack the fibres. It also randomly
cleaves the starch into dextrins that are water soluble
and can be removed by washing. The use of a-amylases
in warp sizing of textile fibres for manufacturing fibres
with great strength has been reported [141].
8.4. Paper industry
The use of a-amylase for the production of low
viscosity, high molecular weight starch for coating of
paper is reported [142]. The use of amylases in the pulp
and paper industry is in the modification of starches for
coated paper. As for textiles, sizing of paper is
performed to protect the paper against mechanical
damage during processing. It also improves the quality
of the finished paper. The size enhances the stiffness and
strength in paper. It also improves the erasibilty and is a
good coating for the paper. Starch is also a good sizing
agent for the finishing of paper. Starch is added to the
paper in the size press and paper picks up the starch by
passing through two rollers that transfer the starch
slurry. The temperature of this process lies in the range
of 45 /60 8C. A constant viscosity of the starch is
required for reproducible results at this stage. The mill
also has the flexibility of varying the starch viscosity for
different paper grades. The viscosity of the natural
starch is too high for paper sizing and is adjusted by
partially degrading the polymer with a-amylases in a
batch or continuous processes. The conditions depend
upon the source of starch and the a-amylase used [143].
A number of amylases exist for use in the paper
ARTICLE IN PRESS
industry, which include Amizyme (PMP Fermentation

Products, Peoria, USA), Termamyl , Fungamyl,
BAN (Novozymes, Denmark) and a-amylase
G9995 (Enzyme Biosystems, USA).
13
developed [96]. 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
[150].
8.5. Detergent applications

Enzymes now comprise as one of the ingredients of
modern compact detergents. The main advantage of
enzyme application in detergents is due to much milder
conditions than with enzyme free detergents. The early
automatic dishwashing detergents were very harsh,
caused injury when ingested and were not compatible
with delicate china and wooden dishware. This forced
the detergent industries to search for milder and more
efficient solutions [144]. Enzymes also allow lowering of
washing temperatures. a-Amylases have been used in
powder laundry detergents since 1975. Nowadays, 90%
of all liquid detergents contain a-amylase [145] and the
demand for a-amylases for automatic dishwashing
detergents is growing. One of the limitations of aamylases in detergents is that the enzyme shows
sensitivity to calcium and stability is severely compromised in a low calcium environment. In addition, most
wild-type a-amylases are sensitive to oxidants which are
generally a component of detergent formulations. Stability against oxidants in household detergents was
achieved by utilising successful strategies followed with
other enzymes such as protease. Recently scientists from
the two major detergent enzyme suppliers Novozymes
and Genencore International have used protein engineering to improve the bleach stability of the amylases
[146 /148]. They independently replaced oxidation sensitive amino acids with other amino acids. The replacement of met at position 197 by leu in B. licheniformis
amylase resulted in an amylase with improved resistance
against oxidative compounds. This improved oxidation
stability resulted in better storage stability and performance of the mutant enzyme in the bleach containing
detergent formulations. Genencore International and
Novozyme have introduced these new products in the
market under the trade names Purafect OxAm and
Duramyl , respectively.
8.6. 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 a-amylase for the Ciba Corning Express
clinical chemistry system has been described [149]. A
process for the detection of higher oligosaccharides,
which involved the application of amylase was also
9. Conclusions
As evident from the foregoing review, amylases are
among the most important enzymes used in industrial
processes. Although, the use of amylases, a-amylases in
particular, in starch liquefaction and other starch based
industries has been prevalent for many decades and a
number of microbial sources exist for the efficient
production of this enzyme, the commercial production
of this enzyme has been limited to only a few selected
strains of fungi and bacteria. Moreover, the demand for
these enzymes is further limited with specific applications as in the food industry, wherein fungal a-amylases
are preferred over other microbial sources due to their
more accepted GRAS status. Structural conformation
plays an important role on amylase activity [151].
Further there arises a need for more efficient a-amylases
in various sectors, which can be achieved either by
chemical modification of the existing enzymes or
through protein engineering. In the light of modern
biotechnology, a-amylases are now gaining importance
in biopharmaceutical applications. Still, their application in food and starch based industries is the major
market and thus the demand of a-amylases would
always be high in these sectors.
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Process Biochemistry 00 (2003) 1 /18

Microbial a-amylases: a biotechnological perspective

* Corresponding author. Tel.: /91-11-2611-1933; fax: /91-112688-5270.

This was followed by several reports of digestive

R. Gupta et al. / Process Biochemistry 00 (2003) 1 /18

lases at length. a-Amylases are one of the most popular

2. Distribution of a-amylase among microorganisms

3. Determination of a-amylase activity

3.3. Degradation of colour-complexed substrate

estimating a-amylase activity which are based on the

4. Physiology of a-amylase production

R. Gupta et al. / Process Biochemistry 00 (2003) 1 /18

4.1.1. Substrate source: induction of a-amylase

[46]. Yeast extract has also been used in conjunction

4.1.3. Role of phosphate

inhibitory to enzyme production by B. amyloliquefaciens

been reported [67]. However, temperatures as high as

5. Fermentation studies on a-amylase production

R. Gupta et al. / Process Biochemistry 00 (2003) 1 /18

[60]. The bulk enzyme activity was nearly 54% greater in

6. Purification of microbial a-amylases

precipitation using ammonium sulphate or organic

7. Biochemical properties of a-amylases

Fungi and yeast

DE52-Cellulose (pH 7.0), 70% (NH4)2SO4, Sephacryl S300, 70% (NH4)2SO4,

Ultrafiltration, 75% ethanol precipitation, Sephadex G-150 (pH 8.0), DEAE

a-Cyclodextrin coupled Sepharose 6B (pH 6.0)

65% (NH4)2S04, CM-Cellulose (pH 6.4)

Thermostabilities have not been estimated defactor in

Carbohydrate moieties raise the molecular weight of

pH optima/stabi- Temperature oplity

Fungi and yeast

Km (0.5 g l 1); Vmax (108.67 [92]

Ag , Cu2 , Fe3 , Hg2 , halides Substrate

Hg2 , Pb2 , maltose

4.8 /5.0/3.5 /6.5

Ag , Cu2 , Hg2 , halides

4.0 /5.0/2.2 /7.0

//40 8C (15 min)

5.0 /6.0/5.0 /8.5

Ag , Cu2 , Fe3 , Hg2 , halides Substrate

Km /7.13, 4.35, 3.12 mM

Higher thermal stability

A. oryzae 245 (ATCC /

R. Gupta et al. / Process Biochemistry 00 (2003) 1 /18

pH optima/stabi- Temperature oplity

Hg2 , Ag2 , Cu2 , Zn2

Cu2 , Mn2 , Zn2

Raw starch digesting en[152]

DTT, Cu2 , Ag2

Km (0.8 g l 1); Kcat (622

End products, G1, G2, G3

Km (0.364 mg ml 1); end

5.0 /5.5/4.0 /7.0

5.0 /6.0/6.0 /7.5

70 /80 8C/(5 days)

Cd2 , Cu2 , Hg2 , Pb2 , Zn2 , Ca2 , Na2 , B.S.A.

Cu2 , Fe3 , Mn2 , Hg2 , Zn2 , Ca2

Hg2 , Fe3 , Al3

End products, G1, G2, G3,

pH optima/stabi- Temperature oplity

Bacillus sp. IMD 434